AU690570B2 - High level expression, purification and refolding of the neisseria meningitidis outer membrane group B porin proteins - Google Patents

Description

WO 95/03413 PCT/US94/08327 HIGH LEVEL EXPRESSION, PURIFICATION ANO REFOLDING OF THE NEISSERIA MENINGITIDIS OUTER MEMBRANE GROUP P PORIN PROTEINS Background of the Invention Field of the Invention The present invention is in the field of recombinant genetics, protein expression, and vaccines. The present invention relates, in particular, to a method of expressing in a recombinant host an outer membrane group B porin protein from Neisseria meningitidis. The invention also relates to a method of purification and refolding of the recombinant protein.

Background Information The outer membranes of Neisseria species much like other Gram negative bacteria are semi-permeable membranes which allow free flow access and escape of small molecular weight substances to and from the periplasmic space of these bacteria but retard molecules of larger size (Heasley, et al., "Reconstitution and characterization of the N.

gonorrhoeae outer membrane permeability barrier," in Genetics and Immunobiology of Neisseria gonorrhoeae, Danielsson and Normark, eds., University of Umea, Umea, pp. 12-15 (1980); Douglas, et al., FEMS Microbiol. Lett. 12:305-309 (1981)). One of the mecnanisms whereby this is accomplished is the inclusion within these membranes of proteins which have been collectively named porins. These proteins are made up of three identical polypeptide chains (Jones, et al., Infect. Immun. 30:773-780 (1980); McDe, Jr. and Johnston, J. Bacteriol. 141:1183-1191 (1980)) and in their native trimer conformation, form water filled, voltagedependent channels within the outer membrane of the bacteria or other membranes to which they have been introduced (Lynch, et al., Biophys. J. 41:62 (1983); Lynch, et al., Biophys. J. 45:104-107 -e II I I SWO 95/03413 PCT/US94/08327 -2- (1984); Young, et al., Proc. Natl. Acad. Sci. USA 80:3831-3835 (1983); Mauro, et al., Proc. Natl. Acad. Sci. USA 85:1071-1075 (1988); Young, et al., Proc. Natl. Acad. Sci. USA 83:150-154 (1986)). Because of the relative abundance of these proteins within the outer membrane, these protein antigens have also been used to subgroup both Neisseria gonorrhoeae and Neisseria meningitidis into several serotypes for epidemiological purposes (Frasch, et al., Rev. Infect.

Dis. 7:504-510 (1985); Knapp, et al., "Overview of epidemiological and clinical applications of auxotype/serovar classification of Neisseria gonorrhoeae," The Pathogenic Neisseriae, Schoolnik, ed., American Society for Microbiology, Washington, pp. 6-12 (1985)). To date, many of these proteins from both gonococci and meningococci have been purified (Heckels, J. Gen. Microbiol. 99:333-341 (1977); James and Heckels, J. Immunol. Meth. 42:223-228 (1981); Judd, Anal. Biochem.

173:307-316 (1988); Blake and Gotschlich, Infect. Immun. 36:277-283 (1982); Wetzler, et al., J. Exp. Med. 168:1883-1897 (1988)), and cloned and sequenced (Gotschlich, et al., Proc. Natl. Acad. Sci. USA 84:8135-8139 (1987); McGuinness, et al., J. Exp. Med. 171:1871-1882 (1990); Carbonetti and Sparling, Proc. Natl. Acad. Sci. USA 84:9084-9088 (1987); Feavers, etal., Infect. Immun. 60:3620-3629 (1992); Murakami, et al., Infect. Immun. 57:2318-2323 (1989); Wolff and Stem, FEMS Microbiol. Lett. 83:179-186 (1991); Ward, et al., FEMS Microbiol. Lett. 73:283-289 (1992)).

The porin proteins were initially co-isolated with lipopolysaccharides. Consequently, the porin proteins have been termed "endotoxin-associated proteins" (Bjornson et al., Infect. Immun. 56:1602- 1607 (1988)). Studies on the wild type porins have reported that full assembly and oligomerization are not achieved unless LPS fiom the corresponding bacterial strain is present in the protein environment -s WO 95/03413 PCT/US94/08327 -3- (Holzenburg et al., Biochemistry 28:4187-4193 (1989); Sen and Nikaido, J. Biol. Chem. 266:11295-11300 (1991)).

The meningococcal porins have been subdivided into three major classifications which in antedated nomenclature were known as Class 1, 2, and 3 (Frasch, et al., Rev. Infect. Dis. 7:504-510 (1985)). Each meningococcus examined has contained one of the alleles for either a Class 2 porin gene or a Class 3 porin gene but not both (Feavers, et al., Infect. Immun. 60:3620-3629 (1992)); Murakami, et al., Infect. Immun.

57:2318-2323 (1989)). The presence or absence of the Class 1 gene appears to be optional. Likewise, all probed gonococci contain only one porin gene with similarities to either the Class 2 or Class 3 allele (Gotschlich, et al., Proc. Natl. Acad. Sci. USA 84:8135-8139 (1987); Carbonettiand Sparling, Proc. Natl. Acad. Sci. USA 84:9084-9088 (1987)).

N. gonorrhoeae appear to completely lack the Class 1 allele. The data from the genes that have been thus far sequenced would suggest that all neisserial porin proteins have at least 70% homology with each other with some variations on a basic theme (Feavers, et al., Infect. Immun.

60:3620-3629 (1992)). It has been suggested that much of the variation seen between these neisserial porin proteins is due to the immunological pressures brought about by the invasion of these pathogenic organisms into their natural host, man. However, very little is known about how the changes in the porin protein sequence effect the functional activity of these proteins.

It has been previously reported that isolated gonococcal porins of the Class 2 allelic type behave electrophysically somewhat differently than isolated gonococcal porins of the Class 3 type in lipid bilayer studies both in regards to their ion selectivity and voltage-dependence (Lynch, E.C., et al., Biophys. J. 41:62 (1983); Lynch, et al., Biophys. J. 45:104- 107 (1984)). Furthermore, the ability of the different porins to enter these '-"IISII~ sl ~CPI~ WO 95/03413 PCT[US94/08327 -4lipid bilayers from intact living bacteria seems to correlate not only with the porin type but also with the neisserial species from which they were donated (Lynch, et al., Biophys. J. 45:104-107 (1984)). It would seem that at least some of these functional attributes could be related to different areas within the protein sequence of the porin. One such functional area, previously identified within all gonococcal Class 2-like proteins, is the site of chymotrypsin cleavage. Upon chymotrypsin digestion, this class of porins lack the ability to respond to a voltage potential and close. Gonococcal Class 3-like porins as well as meningococcal porins lack this sequence and are thus not subject to chymotrypsin cleavage but nonetheless respond by closing to an applied voltage potential (Greco, "The formation of channels in lipid bilayers by gonococcal major outer membrane protein," thesis, The Rockefeller University, New York (1981); Greco, etal., Fed. Proc. 39:1813 (1980)).

The major impediment for such studies has been the ability to easily manipulate the porin genes by modem molecular techniques and obtain sufficient purified protein to carry out the biophysical characterizations of these altered porin proteins. It was early recognized that cloned neisserial porin genes, when expressed in Escherichia coli, were letbal to the host E. coli (Carbonetti and Sparling, Proc. Natl. Acad. Sci. USA 84:9084-9088 (1987); Carbonetti, et al., Proc. Natl. Acad. Sci. USA 85:6841-6845 (1988); Barlow, et al., Infect. Immun. 55:2734-2740 (1987)). Thus, many of these genes were cloned and sequenced as pieces of the whole gene or placed into low copy number plasmids under tight expression control (Carbonetti, et al., Proc. Natl. Acad. Sci. USA 85:6841-6845 (1988)). Under these conditions, even when the entire porin gene was expressed, very little protein accumulated that could be further purified and processed for characterization.

I M WO~ 95/03413 PCT/US94/08327 Another tack to this problem which has met with a modicum of success has been to clone the porin genes into a low copy, tightly controlled expression plasmid, introduce modifications to the porin gene, and then reintroduce the modified sequence back into Neisseria (Carbonetti, et al., Proc. Natl. Acad. Sci. USA 85:6841-6845 (1988)). However, this has also been fraught with problems due to the elaborate restriction endonuclease system present in Neisseria, especially gonococci (Davies, Clin. Microbiol. Rev. 2:S78-S82 (1989)).

The present invention is directed to an approach to overcome these difficulties. The DNA sequence of the mature porin proteins, e.g. class 2 and class 3 as well as fusions thereof, may be amplified using the chromosome of the meningococcal bacteria as a template for the PCR reaction. The amplified porin sequences were ligated and cloned into an expression vector containing the T7 promoter. E. coli strain BL21 lysogenic for the DE3 lambda phage (Studier and Moffatt, J. Mol. Biol.

189:113-130 (1986)), modified to eliminate the ompA gene, was selected as one expression host for the pET-17b plasmid containing the porin gene.

Upon induction, large amounts of the meningococcal porin proteins accumulated within the E. coli without any obvious lethal effects to the host bacterium. The expressed meningococcal porin proteins were extracted and processed through standard procedures and finally purified by molecular sieve chromatography and ion exchange chromatography. As judged by the protein profile from the molecular sieve chromatography, the recombinant meningococcal porins eluted from the column as trimers. To be certain that no PCR artifacts had been introduced into the meningococcal porin genes to allow for such high expression, the inserted PorB gene sequence was determined. Inhibition ELISA assays were used to give further evidence that the expressed recombinant porin proteins had renatured into their natural antigenic and trimer conformation.

I pe I- e -qI Summary of the Invention Porins from different neisserial strains and species have been shown to have differences in both primary amino acid sequence and biophysical characteristics as observed by functional assays. A closer examination of how the changes in the primary amino acid sequence of Neisseria porirn molecules correlate with these observed biophysical changes has been impeded by the ability to easily manipulate the cloned porin genes by modern molecular techniques and then subsequently obtain enough of the expressed modified porin protein to purify and apply to these biophysical functional assays. In this invention, the gene coding for a mature PorB protein, lacking the neisserial promoter and signal sequence, was cloned into the expression plasmid pET-17b and transformed into E. coli. Upon induction, large amounts of the PorB protein was produced.

The expressed porin protein was then manipulated to regenerate its native trimer structure and was then purified. Sufficient purified recombinant porin protein was obtained for further antigenic as well as biophysical characterization. Thus, this sets the stage whereby the

S

l biophysical characterization of these neisserial porin proteins can be 20 examined in more detail.

It is a general object of the invention to provide a method of expressing the meningococcal group B porin protein, in particular, the class 2 and class 3 porin proteins.

It is a specific object of the invention to provide a method for the high level expression of the outer membrane meningococcal group B porin protein or fusion protein thereof in an E. coli host cell having a deleted ompA gene (AompA), comprising: 21/11/97 o X03 i010 z61191 X03401 0 7 transforming the E. coli AompA host cell with a vector comprising a selectable marker and a gene coding for a protein selected from the group consisting of: a mature porin protein, and (ii) a fusion protein which is a mature porin protein fused to amino acids 1 to 20 or 1 to 22 of the T7 gene 10 capsid protein; wherein said gene is operably linked to the T7 promoter; growing the transformed host cell in a culture medium containing a selection agent, and o inducing expression of said protein; wherein the protein so expressed comprises more than about 2% of the total protein expressed in the host cell.

It is another specific object of the invention to provide a method of purifying and refolding a meningococcal group B porin protein and fusion protein produced according to the above-described methods.

5It is a further specific object of the invention to provide a vaccine comprising the meningococcal group B porin protein and fusion protein, produced according to the above methods, in an amount effective to elicit 20 protective antibodies in an animal to Neisseria meningitidis; and a pharmaceutically acceptable diluent, carrier, or excipient.

It is another specific object of the invention to provide the abovedescribed vaccine, wherein said meningococcal group B porin protein or fusion protein is conjugated to a Neisseria meningitidis capsular polysa .charide.

0 AsC o I It is a further specific object of the invention to provide a method of preventing bacterial meningitis in an animal comprising administering to the animal the meningococcal group B porin protein or fusion protein-vaccine produced according to the above-described methods.7 raaa a o a a a aa a a a a aao aa aa o a saaaa a a s r a ae r a a a sc 21/11/97 X034 0 10 z61 197 I s ~Cl~i~s~b LCC WO 95/03413 PCT/US94/08327 -8- It is another specific object of the invention to provide a method of preparing a polysaccharide conjugate comprising: obtabiag the abovedescribed meningococcal group B porin proteir or fusion protein; obtaining a polysaccharide from a Neisseria meningitidis organism; and conjugating the meningococcal group B porin protein or fusion protein to the polysaccharide.

It is another specific object of the invention to provide a method of purifying the above-described meningococcal group B porin protein or fusion protein comprising: lysing the transformed E. coli to release the meningococcal group B porin protein or fusion protein as part of insoluble inclusion bodies; washing the inclusion bodies with a buffer to remove contaminating E. coli cellular proteins; resuspending and dissolving the inclusion bodies in an aqueous solution of a denaturant; diluting the resultant solution in a detergent; and purifying the solubilized meningococcal group B porin protein or fusion protein by gel filtration and ion exchange chromatography.

It is another specific object of the invention to provide a method of refolding the above-described meningococcal group B porin protein or fusion protein comprising: lysing the transformed E. coli to release the meningococcal group B porin protein or fusion protein as part of insoluble inclusion bodies; washing the inclusion bodies with a buffer to remove contaminating E. coli cellular proteins; resuspending and dissolving the inclusion bodies in an aqueous solution of a denaturant; diluting the resultant solution in a detergent; and purifying the solubilized meningococcal group B porin. protein or fusion protein by gel filtration to give the refolded protein in the eluant.

It is another specific object of the invention to provide an E. coli strain BL21 (DE3) AompA host cell that contains a vector which comprises a DNA molecule coding for a meningococcal group B porin protein or

I

WO 95/034813 PCT/US94/08327 -9fusion protein, wherein the DNA molecule is operably linked to the T7 promotor of the vector.

It is another specific object of the invention to provide the E coli strain BL21(DE3)AorpA.

Further objects and advantages of the present invention will be clear from the description that follows.

Brief Description of the Drawings Figure 1: A diagram showing the sequencing strategy of the PorB gene. The PCR product described in Example 1 (Materials and Methods section) was ligated into the BamHI-XhoI site of the expression plasmid pET-17b. The initial double stranded primer exteDsion sequencing was accomplished using oligonucleotide sequences directly upstream of the BamHI site and just downstream of the Xhol site within the pET-17b plasmid. Additional sequence data was obtained by making numerous deletions in the 3' end of the gene, using exonuclease III/mung bean nuclease reactions. After religation and transformation back into E. coli, several clones were selected on size of insert and subsequently sequenced This sequencing was always from the 3' end of the gene using an oligonucleotide primer just downstream of the Bpul 1021 site.

Figure 2: A gel electrophoresis showing the products of the PCR reaction (electrophoresed in a 1% agarose using TAE buffer).

Figure 3 (panels and Panel SDS-PAGE analysis of whole cell lysates of E. coli hosting the control pET-17b plasmid without inserts and an E. coli clone harboring pET-17b plasmid containing an insert from the obtained PCR product described in the materials and methods section. Both cultures were grown to an O.D. of 0.6 at 600 nm, IPTG added, and incubated at 37°C for 2 hrs. 1.5 mis of each of the cultures U- l- lsaslls- raF'~-4ia IClsl~BP WO 95/034i3 PCT/US94/08327 were removed, centrifuged, and the bacterial pellet solubilized in 100 ll of SDS-PAGE preparation buffer. Lane A shows the protein profile obtained with 10 p/ from the control sample and Lanes B (5 tl) and C (10 Ali) demonstrate the protein profile of the E. coli host expressing the PorB protein. Panel Western blot analysis of whole cell lysates of E. coli harboring the control pET-17b plasmid without insert after 2 hrs induction with IPTG, Lane A, 20 Al and a corresponding F coli clone containing a porB-pET-17b plasmid, Lane B, 5 Lane C, 10 dp; and Lane D, 20 1.

The monoclonal antibody 4D11 was used as the primary antibody and the western blot developed as described. The pre-stained low molecular weight standards from BRL were used in each case.

Figure 4: The nucleotide sequence (SEQ ID NO. 1) and the translated amino acid sequence (SEQ ID NO. 2) of the mature PorB gene cloned into the expression plasmid pET-17b. The two nucleotides which differ from the previously published serotype 15 PorB are underlined.

Figure 5: A graph showing the Sephacryl S-300 column elution profile of both the wild type Class 3 protein isolated from the meningococcal strain 8765 and the recombinant Class 3 protein produced by BL21(DE3) -AompAE. coli strain hosting the r3pET-17b [is this a typo it does not appear in the Examples] plasmid as monitored by absorption at 280nm and SDS-PAGE analysis. The void volume of the column is indicated by the arrow. Fractions containing the meningococcal porin and recombinant porin as determined by SDS-PAGE are noted by the bar.

Figure 6: A graph showing the results of the inhibition ELISA assays showing the ability of the homologous, wild type (wt) PorB to compete for reactive antibodies in six human immune sera. The arithmetic mean inhibition is shown by the bold line.

Figure 7: A graph showing the results of the inhibition ELISA assays showing the ability of the purified recombinant PorB protein to I L .WO 95/0313 I)CT/US9408327 11 compete for reactive antibodies in six human immune sera. The arithmetic mean inhibition is shown by the bold line.

Figure 8: A graph showing a comparison of these two mean inhibitions obtained with the wt and recombinant PorB protein.

Figure 9A and 9B: The nucleotide sequence (SEQ ID NO. 3) and the translated amino acid sequence (SEQ ID NO. 4) of the mature class II porin gene cloned into the expression plasmid pET-17b.

Figure 10A and 10B: The nucleotide sequence (SEQ ID NO. and the translated amino acid sequence (SEQ ID NO. 6) of the fusion class II porin gene cloned into the expression plasmid pET-17b.

Figure 11 (panels A and Panel A depicts the restriction map of the pET-17b plasmid. Panel B depicts the nucleotide sequence (SEQ ID NO. 7 AND SEQ ID NO. 9) between the BglII and XhoI sites of pET-17b.

The sequence provided by the plasmid is in normal print while the sequence inserted from the PCR product are identified in bold print. The amino acids (SEQ ID NO. 8 AND SEQ ID NO. 10) which are defived from the plasmid are in normal print while the amino acids from the insert are in bold. The arrows demarcate where the sequence begins to match the sequence in Figure 4 and when it ends.

Detailed Description of the Invention Unlike the porin proteins of E. coli and a few other gram negative bacteria, relatively little is known how changes in the primary sequence of porins from Neisseria effect their ion selectivity, voltage dependence, and other biophysical functions. Recently, the crystalline structure of two E. coli porins, OmpF and PhoE, were solved to 2.4A and respectively (Cowan, et al., Nature 358:727-733 (1992)). Both of these E. coli porins have been intensively studied owing to their unusual C dlL~lsl~ 16 7y WO 95/03413 PCT/VXUS94/08327 12stability and ease with which molecular genetic manipulations could be accomplished. The data obtained for the genetics of these two porins correlated well with the crystalline structure. Although it has been shown in several studies, using monoclonal antibodies to select neisserial porins, that the surface topology of Neisseria closely resembles that of these two E. coli porins (van der Ley, et al., Infect. Immun. 59:2963-2971 (1991)), almost no information is available about how changes in amino acid sequences in specific areas of the neisserial porins effect their biophysical characteristics, as had been done with the E. coli porins (Cowan, et al., Nature 358:727-733 (1992)).

Two of the major problems impeding this research are: the inability to easily manipulate Neisseria genetically by modem molecular techniques and the inability to express sufficient quantities of neisserial porins in E. coli for further purification to obtain biophysical and biochemical characterization data. In fact, most of the DNA sequence data on gonococcal and meningococcal porins have been obtained by cloning overlapping pieces of the porin gene and then reconstructing the information to reveal the entire gene sequence (Gotschlich, et al., Proc. Natl. Acad. Sci. USA 84:8135-8139 (1987); Murakami, et al., Infect. Immun. 57:2318-2323 (1989)). Carbonetti et al. were the first to clone an entire gonococcal porin gene into E. coli using a tightly controlled expression plasmid. The results of these studies showed that when the porin gene was induced, very little porin protein accumulated and the expression of this protein was lethal to the E. coli (Carbonetti and Sparling, Proc. Natl. Acad. Sci. USA 84:9084-9088 (1987)). In additional studies, Carbonetti et al. (Proc. Natl. Acad. Sci. USA 85:6841-6845 (1988)) did show that alterations in the gonococcal porin gene could be made in this system in E. coli and then reintroduced into gonococci. However, the ease with which one can make these manipulations and obtain enough porin ~Plls~ Is =_1 WO 95/03413 PCT/US94/08327 13 protein for further biochemical and biophysical characterization seems limited.

Feavers et al. have described a method to amplify, by PCR, neisserial porin genes from a wide variety of sources using two synthesized oligonucleotides to common domains at the 5' and 3' ends of the porin genes respectively (Feavers, LM., et al., Infect. Immun. 60:3620-3629 (1992)). The oligonucleotides were constructed such that the amplified DNA could be forced cloned into plasmids using the restriction endonucleases BglI and Xhol.

Using the Feavers et al. PCR system, the DNA sequence of the mature PorB protein from meningococcal strain 8765 serotype 15 was amplified and ligated into the BamHI-XhoI site of the T7 expression plasmid pET-17b. This placed the mature PorB protein sequence in frame directly behind the T7 promoter and 20 amino acids of the 410 protein including the leader sequence. Upon addition of IPTG to a culture of E. coli containing this plasmid, large amounts of PorB protein accumulated within the bacteria. A complete explanation for why this construction was non-lethal to the E. coli and expressed large amount of the porin protein, await further studies. However, one possible hypothesis is that by replacing the neisserial promoter and signal sequence with that of the T7 and 410 respectively, the porin product was directed to the cytoplasm rather than toward the outer membrane. Henning and co-workers have reported that when E. coli OmpA protein and its fragments are expressed, those products which are found in the cytoplasm are less toxic than those directed toward the periplasmic space (Klose, et al., J. Biol. Chem.

263:13291-13296 (1988); Klose, et al., J. Biol. Chem. 263:13297- 13302 (1988); Freudl, et al., J. Mol. Biol. 205:771-775 (1989)).

Whatever the explanation, once the PorB protein was expressed, it was easily isolated, purified and appeared to reform into trimers much like the II I WO 95/03413 f3PCT S94/0832'7 14native porin. The results of the inhibition ELISA data using human immune sera suggests that the PorB protein obtained in this fashion regains most if not all of the antigenic characteristics of the wild typ PorB protein purified from meningococci. This expression system lends itself to the easy manipulation of the neisserial porin gene by modern molecular techniques.

In addition, this system allows one to obtain large quantities of pure porin protein for characterization. In addition, the present expression system allows the genes from numerous strains of Neisseria, both gonococci and meningococci, to be examined and characterized in a similar manner.

Thus, the present invention relates to a method of expressing an outer membrane meningococcal group B porin protein, in particular, the class 2 and class 3 porin proteins.

In one embodiment, the present invention relates to a method of expressing the outer membrane meningococcal group B porin protein in E. coli comprising: transforming E. coli by a vector comprising a selectable marker and a gene coding for a plutein selected from the group consisting of: a mature porin protein, and (ii) a fusion protein comprising a mature porin protein fused to amino acids 1 to 20 or 22 of the T7 gene 0b10 capsid protein; wherein said gene is operably linked to the T7 promoter; growing the transformed E. coli in a culture media containing a selection agent, and inducing expression of said protein; wherein the protein so produced comprises more than about 2 of the total protein expressed in the E. coli.

I ill I e WO 95/03413 PCTIUS94/08327 15 In a preferred embodiment, the meningococcal group B porin protein or fusion protein expressed comprises more than about 5% of the total proteins expressed in E. coli. In another preferred embodiment, the meningococcal group B porin protein or fusion protein expressed comprises more than about 10% of the total proteins expressed in E. coli. In yet another preferred embodiment, the meningococcal group B porin protein or fusion protein expressed comprises more than about 30% of the total proteins expressed in E. coli.

Examples of plasmids which contain the T7 inducible promotor include the expression plasmids pET-17b, pET-11a, pET-24a-d(+) and pET-9a, all of which are commercially available from Novagen (565 Science Drive, Madison, WI 53711). These plasmids comprise, in sequence, a T7 promoter, optionally a lac operator, a ribosome binding site, restriction sites to allow insertion of the structural gene and a T7 terminator sequence. See, the Novagen catalogue, pages 36-43 (1993).

In a preferred embodiment, E. coli strain BL21 (DE3) AompA is employed. The above mentioned plasmids may be transformed into this strain or the wild-type strain BL21(DE3). E. coli strain BL21 (DE3) AompA is preferred as no OmpA protein is produced by this strain which might contaminate the purified porin protein and create undesirable immunogenic side effects.

The transformed E. coli are grown in a medium containing a selection agent, e.g. any f-lactam to which E. coli is sensitive such as ampicillin. The pET expression vectors provide selectable markers which confer antibiotic resistance to the transformed organism.

High level expression of meningococcal group B porin protein can be toxic in E. coli. Surprisingly, the present invention allows E. coli to express the protein to a level of at least almost 30% and as high as of the toU.. r- ular proteins.

L-L-e Q WO 9/0413 0CTJUS94/83 Z -16- In another preferred emiodiment, the present invention relates to a vaccine comprising the outer membrane meningococcal group B porin protein or fusion protein thereof, produced according to the abovedescribed methods, together with a pharmaceutically acceptable diluent, carrier, or excipient, wherein the vaccine may be administered in an amount effective to elicit protective antibodies in an animal to Neisseria meningitidis. In a preferred embodiment, the animal is selected from the group consisting of humans, cattle, pigs, sheep, and chickens. In another preferred embodiment, the animal is a human.

In another preferred embodiment, the present invention relates to the above-described vaccine, wherein said outer membrane meningococcal group B porin protein or fusion protein thereof is conjugated to a meningococcal group B capsular polysaccharide Such capsular polysaccharides may be prepared as described in Ashton, F.E. et al., Microbial Pathog. 6:455-458 (1989); Jennings, H.J. et al., J. Immunol.

134:2651 (1985); Jennings, H.J. et al., J. Immunol. 137:1708-1713 (1986); Jennings, H.J. et al., J. Immunol. 142:3585-3591 (1989); Jennings, H.J., "Capsular Polysaccharides as Vaccine Candidates," in Current Topics in Microbiology and Immunology, 150:105-107 (1990); the contents of each of which are fully incorporated by reference herein.

Preferably, the CP is isolated according to Frasch, C.E., "Production and Control of Neisseria meningitidis Vaccines" in Bacterial Vaccines, Alan R. Liss, Inc., pages 123-145 (1990), the contents of which are fully incorporated by reference herein, as follows: Grow organisms in modified Franz medium 10 to 20 hrs J Heat kill, 55 C, 10 min Remove inactivated cells by centrifugation I Add Cetavlon to 0.1% Precipitate CP from culture broth II -a r re WO 95/03413 PCT/US94/08327 17- 1 Add calcium chloride to 1 M Dissolve CP then centrifuge to remove cellular debris I Add ethyl alcohol to 25 Remove precipitated nucleic acids by centrifugation 1 Add ethyl alcohol to Precipitate crude CP and remove alcohol The crude CP is then further purified by gel filtration chromatography after partial depolymerization with dilute acid, e.g.

acetic acid, formic acid, and trifluoroacetic acid (0.01-0.5 to give a mixture of polysaccharides having an average molecular weight of 12,000- 16,000. The CP is then N-deacetylated with borohydride and Npropionylated to afford N-Pr GBMP. Thus, the CP tft be employed in the conjugate vaccines of the present invention may CP frnagments, Ndeacylated CP and fragments thereof, as well as N-Pr CP and fragments thereof, so long as they induce active immunity when employed as part of a CP-porin protein conjugate (see the Examples).

In a further preferred embodiment, the present invention relates to a method of preparing a polysaccharide conjugate comprising: obtaining the above-described outer membrane meningococcal group B porin protein or fusion protein thereof; obtaining a CP from a Neisseria meningitidis organism; and conjugating the protein to the CP.

The conjugates of the invention may be formed by reacting the reducing end groups of the CP to primary amino groups of the porin by reductive amination. The reducing groups may be formed by selective hydrolysis or specific oxidative cleavage, or a combination of both.

Preferably, the CP is conjugated to the porin protein by the method of Jennings et al., U.S. Patent No. 4,356,170, the contents of which are fully p 9b IWDIIIII~ sl~* ICI)R L IIPIIYI~~*I WO 95/03413 PC T/VJS94I0327 -18incorporated by reference herein, which involves controlled oxidation of the CP with periodate followed by reductive amination with the porin protein.

The vaccine of the present invention comprises the meningococcal group B porin protein, fusion protein or conjugate vaccine in an amount effective depending on the route of administration. Although subcutaneous or intramuscular routes of administration are preferred, the meningococcal group B porin protein, fusion protein or vaccine of the present invention can also be administered by an intraperitoneal or intravenous route. One skilled in the art will appreciate that the amounts to be administered for any particular treatment protocol can be readily determined without undue experimentation. Suitable amounts might be expected to fall within the range of 2 micrograms of the protein per kg body weight to 100 micrograms per kg body weight.

The vaccine of the present invention may be employed in such forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions. Any inert carrier is preferably used, such as saline, phosphate-buffered saline, or any such carrier in which the meningococcal group B porin protein, fusion protein or conjugate vaccine have suitable solubility properties. The vaccines may be in the form of single dose preparations or in multi-dose flasks which can be used for mass vaccination programs. Reference is made to Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, Osol (ed.) (1980); and New Trends and Developments in Vaccines, Voller et al.

University Park Press, Baltimore, MD (1978), for methods of preparing and using vaccines.

The meningococcal group B porin protein, fusion protein or conjugate vaccines of the present invention may further comprise adjuvants which enhance production of porin-specific antibodies. Such adjuvants include, but are not limited to, various oil formulations such as Freund's Ilol dr I WO 95/03413 WCT/(189408327 -19complete adjuvant (CFA), stearyl tyrosine (ST, see U.S. Patent No.

4,258,029), the dipeptide known as MDP, saponin, aluminum hydroxide, and lymphatic cytokine.

Freund's adjuvant is an emulsion of mineral oil and water which is mixer with the immunogenic substance. Although Freund's adjuvant is powerful, it is usually not administered to humans. Instead, the adjuvant alum (aluminum hydroxide) or ST may be used for administradon to a human. The meningococcal group B porin protein or a conjugate vaccine thereof may be absorbed onto the aluminum hydroxide from which it is slowly released after injection. The meningococcal group B porin protein or conjugate vaccine may also be encapsulated within liposomes according to Fullerton, U.S. Patent No. 4,235,877.

In another preferred embodiment, the present invention relates to a method of preventing bacterial meningitis in an animal comprising administering to the animal the meningococcal group B porin protein, fusion protein or conjugate vaccine produced according to methods described in an amount effective to prevent bacterial meningitis.

In a further embodiment, the invention relates to a method of purifying the above-described outer membrane meningococcal group B porin protein or fusion protein comprising: lysing the transformed E. coli to release the meningococcal group B porin protein or fusion protein as part of insoluble inclusion bodies; washing the inclusion bodies with a buffer to remove contaminating E. coli cellular proteins; resuspending and dissolving the inclusion bodies in an aqueous solution of a denaturant; diluting the resultant solution in a detergent; and purifying the solubilized meningococcal group B porin protein by gel filtration.

The lysing step may be carried out according to any method known to those of ordinary skill in the art, e.g. by sonication, enzyme digestion, osmotic shock, or by passing through a mull press.

8 WO 95/03413 WO TIJS94/08327 The inclusion bodies may be washed with any buffer which is capable of solubilizing the E, coli cellular proteins without solubilizing the inclusion bodies comprising the meningococcal group B porin protein.

Such buffers include but are not limited to TEN buffer (50 mM Tris HC1, 1 mM EDTA, 100 mM NaC1, pH Tricine, Bicine and HEPES.

Denaturants which may be used in the practice of the invention include 2 to 8 M urea or about 2 to 6 M guanidine HC1, more preferably, 4 to 8 M urea or about 4 to 6 M guanidine HC1, and most preferably, about 8 M urea or about 6 M guanidine HC1).

Examples of detergents which can be used to dilute the solubilized meningococcal group B porin protein include, but are not limited to, ionic detergents such as SDS and cetavlon (Calbiochem); non-ionic detergents such as Tween, Triton X, Brij 35 and octyl glucoside; and zwitterionic detergents such as 3,14-Zwittergent, empigen BB and Champs.

Finally, the solubilized outer membrane meningococcal group B porin protein may be purified by-gel filtration to separate the high and low molecular weight materials. Types of filtration gels include but are not limited to Sephacryl-300, Sepharose CL-6B, and Bio-Gel A-1.5m. The column is eluted with the buffer used to dilute the solubilized protein. The fractions containing the porin or fusion thereof may then be identified by gel electrophoresis, the fractions pooled, dialyzed, and concentrated.

Finally, substantially pure 95 porin protein and fusion protein may be obtained by passing the concentrated fractions through a Q sepharose high performance column.

In another embodiment, the present invention relates to expression of the meningococcal group B porin protein gene which is part of a vector which comprises the T7 promoter, which is inducible. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. The T7 promoter is inducible by the addition of i- ~s illh*t~l~l~llg~l~ WO 95/03413 4IT1t1594lO8317 -21 isopropyl O-D-thiogalactopyranoside (IPTG) to the culture medium.

Alternatively, the Tac promotor or heat shock promotor may be employed.

Preferably, the menigococcal group B porin protein gene is expressed from the pET-17 expression vector or the pET-lla expression vector, both of which contain the T7 promoter.

The cloning of the meningococcal group B porin protein gene or fusion gene into an expression vector may be carried out in accordance with conventional techniques, including blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline pbosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Reference is made to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press (1989), for general methods of cloning.

The meningococcal group B porin protein and fusion protein expressed according to the present invention must be properly refolded in order to achieve a structure which is immunologically characteristic of the native protein. In yet another embodiment, the present invention relates to a method of refolding the above-described outer membrane protein and fusion protein comprising: lysing the transformed E. coli to release the meningococcal group B porin protein or fusion protein as part of insoluble inclusion bodies; washing the inclusion bodies with a buffer to remove contaminating E. coli cellular proteins; resuspending and dissolving the inclusion bodies in an aqueous solution of a denaturant; diluting the resultant solution in a detergent; and purifying the solubilized meningococcal group B porin protein or fusion protein by gel filtration to give the refolded protein in the eluant. Surprisingly, it has been discovered that the folded trimeric meningococcal group B class 2 and class 3 porin I _I I, I WO 95/03413 pC'/U/S94/08327 -22proteins qnd fusion proteins are obtained directly in the eluant from the gel filtration column.

In another preferred embodiment, the present invention relates to a substantially pure refolded outer membrane meningococcal group B porin protein and fusion protein produced according to the above-described methods. A substantially pure protein is a protein that is generally lacking in other cellular Neisseria meningitidis components as evidenced by, for example, electrophoresis. Such substantially pume proteins have a purity of as measured by densitometry on an electrophoretic gel after staining with Coomassie blue or silver stains.

The following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in this art which are obvious to those skilled in the art are within the spirit and scope of the present invention.

Examples Example 1. Cloning of the Class 3 Porin Protein from Group B Neisseria meningitidis Materials and Methods Organisms: The Group B Neisseria meningitidis strain 8765 (B:15:P1,3) was obtained from Dr. Wendell Zollinger (Walter Reed Army Institute for Research) and grown on agar media previously described (Swanson, Infect. Immun. 21:292-302 (1978)) in a candle extinction jar in an incubator maintained at 30°C. Escherichia coli strains DME558 (from the collection of S. Benson; Silhavy, T.J. et al., "Experiments with Gene Fusions," Cold Spring Harbor Laboratory, Cold Spring Harbor,

~C

WO 95/03413 PCT/JS94/08327 23 1984), BRE51 (Bremer, E. et al., FEMSMicrobiol. Lett. 33:173-178 (1986)) and BL21(DE3) were grown on LB agar plates at 37 0

C.

P1 Transduction: A Plvr lysate of E. coli strain DME558 was used to transduce a tetracycline resistance marker to strain BRE51 (Bremer, E., et al., FEMS Microbiol. Lett. 33:173-178 (1986)) in which the entire ompA gene had been deleted (Silhavy, et al., Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984)).

Strain DME558, containing the tetracycline resistance marker in close proximity of the ompA gene, was grown in LB medium until it reached a density of approximately 0.6 OD at 6(0 nm. One tenth of a milliliter of M CaC1 was added to the 10 ml culture and 0.1 ml of a solution containing 1 x 10 9 PFU of Plvir. The culture was incubated for 3 hours at 37°C. After this time, the bacterial cell density was visibly reduced.

ml of chloroform was added and the phage culture stored at 4 0

C.

Because typically 1-2% of the E. coli chromosome can be packaged in each phage, the number of phage generated covers the entire bacterial host chromosome, including the tetracycline resistance marker close to the ompA gene.

Next, strain BRE51, which lacks the ompA gene, was grown in LB medium overnight at 37 0 C. The overnight culture was diluted 1:50 into fresh LB and grown for 2 hr. The cells were removed by centrifugation and resuspended in MC salts. 0.1 ml of the bacterial cells were mixed with 0.05 of the phage lysate described above and incubated for 20 min. at room temperature. Thereafter, an equal volume of 1 M sodium citrate was added and the bacterial cells were plated out onto LB plates containing 12.5 1 g/ml of tetracycline. The plates were incuoated overnight at 37°C.

Tetracycline resistant (12 tig/ml) transductants were screened for lack of OmpA protein expression by SDS-PAGE and Western Blot analysi as described hb-w. The bacteria resistant to the antibiotic have the LL I I PP~ WO 95/03413 PCT/US9408327 24 tetracycline resistance gene integrated into the chromosome very near where the ompA gene had been deleted from this strain. One particular strain was designated BRE-T a A second round of phage production was then carried out with the strain BRE-TR, using the same method as described above. Representatives of this phage population contain both the tetracycline resistance gene and the OmpA deletion. These phage were then collected and stored. These phage were then used to infect E. coli BL21(DE3). After infection, the bacteria contain the tetracycline resistance marker. In addition, there is a high probability that the OmpA deletion was selected on the LB plates containing tetracycline.

Colonies of bacteria which grew on the plates were grown up separately in LB medium and tested for the presence of the OmpA protein.

Of those colonies selected for examination, all lacked the OmpA protein as judged by antibody reactivity on SDS-PAGE western blots.

SDS-PAGE and Western Blot: The SDS-PAGE was a variation of Laemmli's method (Laemmli, Nature 227:680-685 (1970)) as described previously (Blake and Gotschlich, J. Exp. Med. 159:452-462 (1984)). Electrophoretic transfer to Immobilon P (Millipore Corp. Bedford, MA) was performed according to the methods of Towbin et al.. (Towbin, et al., Proc. Natl. Acad. Sci. USA 76:4350-4354 (1979)) with the exception that the paper was first wetted in methanol. The Western blots were probed with phophatase conjugated reagents (Blake, et a., Analyt. Biochem. 136:17.-179 (1984)).

Po ymerase Chain Reaction: The method described by Feavers et al. (Feavers, et al., Infect. Immun. 60:3620-3629 (1992)) was used to amplify the gene encoding the PorB. The primers selected were primers 33 (SEQ ID NO. 11) (GGG GTA GAT CTG CAG GTT ACC TTG TAC GGT ACA ATT AAA GCA GGC GT) and 34 (SEQ ID NO. 12) (GGG I a~ WO 95/0413 W91Cr/1I$94/08321 GGG GTG ACC CTC GAG TTA GAA TTT GTG ACG CAG ACC AAC) as previously described (Feavers, et al., Infect. Immun. 60:3620-3629 (1992)). Briefly, the reaction components were as follows: Meningococcal strain 8765 chromosomal DNA (100 1 Al; 5' and 3' primers (1 AM) 2 ,l each; dNTP (10 mM stocks), 4 pl each; 10 X PCR reaction buffer (100 mM Tris HCI, 500 mM KC1, pH 10 Al; 25 mM MgC12, 6 l; double distilled H 2 0, 62 Al; and Taq polymerase (Cetus Corp., 5 u/Al), 1 Al. The reaction was carried out in a GTC-2 Genetic Thermocycler (Precision Inst. Inc, Chicago, IL) connected to a Lauda 4/K methanol/water cooling system (Brinkman Instruments, Inc., Westbury, NY) set at 0*C.

The thermocycler was programmed to cycle 30 times through: 94 0 C, 2 min.; 40 0 C, 2 min.; and 72°C, 3 min. At the end of these 30 cycles, the reaction was extended at 72*C for 3 min and finally held at 4*C until readied for analysis on a 1% agarose gel in TAE buffer as described by Maniatis (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,-Cold Spring Harbor, NY (1982)).

Subcloning of the PCR product: The pET-17b plasmid (Novagen, Inc.) was used for subcloning and was prepared by double digesting the plasmid with the restriction endonucleases BamHI and Xhol (New England Biolabs, Inc., Beverly, MA). The digested ends were then dephosph'.rylated with calf intestinal alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN). The digested plasmid was then analyzed on a 1% agarose gel, the cut plasmid removed, and purified using the GeneClean kit (Biol01, La Jolla, CA). The PCR product was prepared by extraction with phenol-chloroform, chloroform, and finally purified using the GeneClean Kit (BiolOl). The PCR product was digested with restriction endonucleases BglII and Xhol (New England Biolabs, Inc.).

The DNA was then extracted with phenol-chloroform, precipitated by adding 0.1 volumes of 3 M sodium acetate, 5 Al glycogen (20 Ag/pl), and I- WO 95/03413 PCT/US94/08327 -26volumes of ethanol. After washing the DNA with 70% ethanol (vol/vol), it was redissolved in TE buffer. The digested PCR product was ligated to the double digested pET-17b plasmid described above using the standard T4 ligase procedure at 16 0 C overnight (Current Protocols in Molecular Biology, John Wiley Sons, New York (1993)). The ligation product was then transformed into the BL21 (DE3)-AompA described above which were made competent by the method of Chung et al. (Chung, C.T., etal., Proc. Natl. Acad. Sci. USA 86:2172-2175 (1989)). The transformants were selected on LB plates containing 50 /g/ml carbenicillin and 12jg/ml tetracycline. Several transformants were selected, cultured in LB both containing carbenicillin and tetracycline for 6 hours at 30 0 C, and plasmid gene expression inducted by the addition of IPTG. The temperature was raised to 37 C and the cultures continued for an additional 2 hrs. The cells of each culture were collected by centrifugation, whole cell lysates prepared, and analyzed by SDS-PAGE and Western Blot using a monoclonal antibody (4D11) which reacts with all neisserial porins.

Nucleotide Sequence Analysis: The nucleotide sequences of the cloned Class 3 porin gene DNA were determined by the dideoxy method using denatured double-stranded plasmid DNA as the template as described (Current Protocols in Molecular Biology, John Wiley Sons, New York (1993)). Sequenase I kits (United States Biochemical Corp., Cleveland, OH) were used in accordance with the manufacturer's instructions. The three synthesized oligonucleotide primers (Operon Technologies, Inc., Alameda, CA) were used for these reactions. One for the 5' end(SEQ ID NO. 13) which consisted of 5'TCAAGCTTGGTACCGAGCTC and two for the 3' end, (SEQ ID NO. 14) 5'TTTGTTAGCAGCCGGATCTG (SEQ 1D NO. 15) and 5' CTCAAGACCCGTTTAGAGGCC. Overlapping, nested deletions were made by linearizing the plasmid DNA by restriction endonuclease Bpul 1021 and the ends blunt.d by the addition of Thio-dNTP ~-~YO-PIIIB i II ~p ~I Is II WO 95/03413 PCT/US94/08327 -27and Klenow polymerase (Current Protocols in Molecular Biology, John Wiley Sons, New York (1993)). The linearized plasmid was then cleaved with restriction endonuclease Xhol and the exoII/Mung bean nuclease deletion kit used to make 3' deletions of the plasmid (Stratagene, Inc., La Jolla, CA) as instructed by the supplier. A map of this strategy is shown in Figure 1.

Expression and purification of the PorB gene product: Using a sterile micropipette tip, a single colony of the BL21 (DE3)-AompA containing the PorB-pET-17b plasmid was selected and inoculated into ml of LB broth containing 50 pg/ml carbenicillin. The culture was incubated overnight at 30°C while shaking. The 10 ml overnight culture was then sterily added to 1 liter of LB broth with the same concentration of carbenicillin, and the culture continued in a shaking incubator at 37 0

C

until the ODoo reached 0.6-1.0. Three mis of a stock solution of IPTG (100 mM) was added to the culture and the culture incubated for an additional 30 min. Rifampicin was then added (5.88 ml of a stock solution; 34 mg/ml in methanol) and the culture continued for an additional 2 hrs.

The cells were harvested by centrifugation at 10,000 rpm in a GS3 rotor for 10 min and weighed. The cells were thoroughly resuspended in 3 ml of TEN buffer (50 mM Tris HC1, 1 mM Tris HC1, 1 mM EDTA, 100 mM NaCI, pH 8.0) per gram wet weight of cells. To this was added 8 cl of PMSF stock solution (50 mM in anhydrous ethanol) and 80 1 l of a lysozyme stock solution (10 mg/ml in water) per gram wet weight of cells.

This mixture was stirred at room temperature for 20 min. While stirring, 4 mg per gram wet weight of cells of deoxycholate was added. The mixture was placed in a 37 0 C water bath and stirred with a glass rod.

When the mixture became viscous, 20 /l of DNase I stock solution (1 mg/ml) was added per gram weight wet cells. The mixture was then removed from the water bath and left at room temperature until the solution i, WO 95/03413 PCT/US94/08327 -28was no longer viscous. The mixture was then centrifuged at 15,000 rpm in a SS-34 rotor for 20 miil at 4°C. The pellet was retained and thoroughly washed twice with TEN buffer. The pellet was then resuspended in freshly prepared TEN buffer containing 0.1 mM PMSF and 8 M urea and sonicated in a bath sonicator (Heat Systems, Inc., Plainview, NY). The protein concentration was determined using a BCA kit (Pierce, Rockville, IL) and the protein concentration adjusted to less than 10 mg/ml using the TEN-urea buffer. The sample was then diluted 1:1 with 10% (weight/vol) Zwittergent 3,14 (CalBiochem, La Jolla, CA), sonicated, and loaded onto a Sephacryl S-300 molecular sieve column. The Sephacryl S-300 column cm x 200 cm) had previously equilibrated with 100 mM Tris HC1, 200 mM NaC1, 10 mM EDTA, 0.05% Zwittergent 3,14, and 0.02% azide, pH The column flow rate was adjusted to 8 ml/hr and 10 ml fractions were collected. The OD2o of each fraction was measured and SDS-PAGE analysis performed on protein containing fractions.

Inhibition ELISA Assays: Microtiter plates (Nunc-Immuno Plate IIF, Nunc, Inc., Naperville, IL) were sensitized by adding 0.1 ml per well of porB (2 gg/ml) purified from the wild type strain 8765, in 0.1 M carbonate buffer, pH 9.6 with 0.02% azide. The plates were incubated overnight at room temperature. The plates were washed five times with 0.9% NaC1, 0.05% Brij 35, 10 mM sodium acetate pH 7.0, 0.02% azide.

Human immune sera raised against the Type 15 Class 3 PorB protein was obtained from Dr. Phillip 0. Livingston, Memorial-Sloan Kettering Cancer Center, New York, N.Y. The human immune sera was diluted in PBS with 0.5% Brij 35 and added to the plate and incubated for 2 hr at room temperature. The plates were again washed as before and the secondary antibody, alkaline phosphatase conjugated goat anti-human IgG (Tago Inc., Burlingame, CA), was diluted in PBS-Brij, added to the plates and incubated for 1 hr at room temperature. The plates were washed as before sits I I r C C~- I W 9.5103413 PC'/US94/08327 -29and p-nitrophenyl phosphate (Sigma Phosphatase Substrate 104) (1 mg/ml) in 0.1 diethanolamine, 1 mM MgCl, 0.1 mM ZnCI 2 0.02% azide, pH 9.8, was added. The plates were incubated at 37 0 C for 1 h and the absorbance at 405 nm determined using an Elida-5 microtiter plate reader (Physica, New York, NY). Coniuol wells lacked either the primary and/or secondary antibody. This was done to obtain a titer for each human serum which would give a half-maximal reading in the ELISA assay. This titer for each human serum would be used in the inhibition ELISA. The ELISA microtiter plate would be sensitized with purified wild type PorB protein and washed as before. In a separate V-96 polypropylene microtiter plate (Nunc, Inc.), varying amounts of either purified wild type PorB protein or the purified recombinant PorB protein were added in a total volume of pl. The human sera were diluted in PBS-Brij solution to twice their half maximal titer and 75 yl added to each o' the wells containing the PorB or recombinant PorB proteins. This plate was incubated for 2 hr at room temperature and centrifuged in a Sorvall RT6000 refrigerated centrifuge, equipped with microtiter plate carriers (Wilmington, DE) at 3000 rpm for min. Avoiding the V-bottom, 100 11 from each well was removed and transferred to the sensitized and washed ELISA microtiter plate. The ELISA plates are incubated for an additional 2 hr, washed, and the conjugated second antibody added as before. The plate is then processed and read as described. The percentage of inhibition is then processed and read as described. The percentage of inhibition is calculated as follows: 1- (ELISA value with either PorB or rPorB protein added) x100 (ELISA value without the porB added) sI SWO 95/03413 1ICTICS904/08327 Results Polymerase Chain Reaction and Subcloning: A method to easily clone, genetically manipulate, and eventually obtain enough pure porin protein from any number of different neisserial porin genes for further antigenic and biophysical characterization 'has been developed. The first step toward this goal was cloning the porin gene from a Neisseria. Using a technique originally described by Feavers, et al. (Feavers, et al., Infect. Immun. 60:3620-3629 (1992)), the DNA sequence of the mature porin protein from a class 3, serotype 15 porin was amplified using the chromosome of meningococcal strain 8765 as a template for the PCR reaction. Appropriate endonuclease restriction sites had been synthesized onto the ends of the oligonucleotide primers, such that when cleaved, the amplified mature porin sequence could be directly ligated and cloned into the chosen expression plasmid. After 30 cycles, the PCR products shown in Figure 2 were obtained. The major product migrated between 900bp and l000bp which was in accord with the previous study (Feavers, et al., Infect. Immun. 60:3620-3629 (1992)). However, a higher molecular weight product was not seen, even though the PCR was conducted under low annealing stringencies (40°C; 50 mM KC1).

To be able to produce large amounts of the cloned porin protein, the tightly controlled expression system of Studier, et al. (Studier and Moffatt, J. Mol. Biol. 189:113-130 (1986)) was employed, which is commercially available through Novagen Inc. The amplified PCR product was cloned into the BamHI-XhoI site of plasmid pET-17b. This strategy places the DNA sequence for the mature porin protein in frame directly behind the T7 promoter, the DNA sequence encoding for the 9 amino acid leader sequence and 11 amino acids of the mature 410 protein. The Studier E.

coli strain BL21 lysogenic for the DE3 lambda derivative (Studier and I a I lI WO 95/03413 PCT/US94/08327 -31 Moffatt, J. Mol. Biol. 189:113-130 (1986)) was selected as the expression host for the pET-17b plasmid containing the porin gene. But because it was thought that the OmpA protein, originating from the E. coli expression host, might tend to co-purify with the expressed meningococcal porin protein, a modification of this strain was made by P1 transduction which eliminated the ompA gene from this strain. Thus, after restriction endonuclease digestion of both the PCR product and the pET-17b vector and ligation, the product was transformed into BL21(DE3)-AompA and transformants selected for ampicillin and tetracycline resistance. Of the numerous colonies observed on the selection plate, 10 were picked for further characterization. All ten expressed large amounts of a protein, which migrated at the approximate molecular weight of the PorB protein, when grown to log phase and induced with IPTG. The whole cell lysate of one such culture is shown in Figure 3a. The western blot analysis with the 4D11 monoclonal antibody further suggested that the protein being expressed was the PorB protein (Figure 3b). As opposed to other studies, when neisserial porins have been cloned and expressed in E. coli, the host bacterial cells showed no signs of any toxic or lethal effects even after the addition of the IPTG. The E. coli cells appeared viable and could be recultured at any time throughout the expression phase.

Nucleotide sequence analysis: The amount of PorB expressed in these experiments was significantly greater than that previously observed and there appeared to be no adverse effects of this expression on the host E. coli. To be certain that no PCR artifacts had been introduced into the meningococcal porin gene to allow for such high expression, the entire porin fusion was sequenced by double stranded primer extension from the plasmid. The results are shown in Figure 4. The nucleotide sequence was identical with another meningococcal serotype 15 PorB gene sequence previously reported by Heckels, et al. (Ward, et al., FEMS I*Cn~R Xl~bC~'Ili C WO 95/03413 I'M'.'US94100327r -32- Microbiol. Lett. 73:283-289 (1992)) with two exceptions which are shown.

These two nucleotide differences each occur in the third position of the codon and would not alter the amino acid sequence of the expressed protein. Thus, from the nucleotide sequence, there did not appear to be any PCR artifact or mutation which might account for the high protein expression and lack of toxicity within the E. coli. Furthermore, this data would suggest that a true PorB protein was being produced.

Purification of the expressed porB gene product: The PorB protein expressed in the E. coli was insoluble in TEN buffer which suggested that when expressed, the PorB protein formed into inclusion bodies. However, washing of the insoluble PorB protein with TEN buffer removed most of the contaminating E. coli proteins. The PorB protein could then be solubilized in freshly prepared 8M urea and diluted into the Zwittergent 3,14 detergent. The final purification was accomplished, using a Sephacryl S-300 molecular sieve column which not only removed the urea but also the remaining contaminating proteins. The majority of the PorB protein eluted from the column having the apparent molecular weight of trimers much like the wild type PorB. The comparative elution patterns of both the wild type and the PorB expressed in the E. coli are shown in Figure 5. It is important to note that when the PorB protein concentration in the 8 M urea was in excess of 10 mg/ml prior to dilution into the Zwittergent detergent, the relative amounts of PorB protein found as trimers decreased and appeared as aggregates eluting at the void volume. However, at protein concentrations below 10 mg/ml in the urea buffer, the majority of the PorB eluted in the exact same fraction as did the wild type PorB. It was also determined using a T7-Tag monoclonal antibody and western blot analysis that the 11 amino acids of the mature T7 capsid protein were retained as the amino terminus. The total yield of the meningococcal porin protein from one liter of E. coli was approximately 50 mg.

~sllsr PI I--~b-41--~BCI SS~rsr WO 95/03413 VCVUS94/0832,7~ -33- Inhibition ELISA Assays. In order to determine if the purified trimeric recombinant PorB had a similar antigenic conformation as compared to the PorB produced in the wild type meningococcal strain 8765, the sera from six patients which had been vaccinated with the wild type meningococcal Type 15 PorB protein were used in inhibition ELISA assays. In the inhibition assay, antibodies reactive to the native PorB were competitively inhibited with various amounts of either the purified recombinant PorB or the homologous purified wild type PorB. The results of the inhibition with the homologous purified PorB of each of the six human sera and the mean inhibition of these sera are shown in Figure 6.

The corresponding inhibition of these sera with the purified recombinant PorB is seen in Figure 6b. A comparison of the mean inhibition from Figure 6 and 7 are plotted in Figure 8. These data would suggest that the antibodies contained in the sera of these six patients found similar epitopes on both the homologous purified wild type PorB and the purified recombinant PorB. This gave further evidence that the recombinant PorB had regained most if not all of the native conformation found in the wild type PorB.

Example 2. Cloning of the Class 2 Porin from Group B Neisseria Meningitiis strain BNCV M986 Genomic DNA was isolated from approximately 0.5g of Group B Neisseria meningitidis strain BNCV M986 (serotype 2a) using previously described methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press (1989)). This DNA then served as the template for two class 2 porin specific oligonucleotides in a standard PCR reaction. These oligonucleotides were designed to be complementary to the 5' and 3' flanking regions of the class 2 porin and to contain EcoRI restriction sites LIL~ tl I WO 95/03413 '11CIVUS94/03327 34 to facilitate the cloning of the fragment. The sequences of the oligonucleotdes were as follows: (SEQ ID NO, 16) AGC 0OC TTO GM TTC CCG GCT GGC TTA AAT TTC 3' (SEQ ID NO. 17) and 5' CAA ACG AAT GAA TTC AAA TAA AAA AGC CTG 3'.

The polymerase chain reaction was then utilized to obtain the class 2 porin.

The reaction conditions were as follows: BNCV M986 genomic DNA 200ng, the two oligonucleotide primers described above at 1 t4M of each, 200 {M of each dNTP, PCR reaction buffer (10 mM Tris HC1, 50 mM KC1, pH 1.5 mM MgCI 2 and 2.5 units of Taq polymerase, made up to 100 /l with distilled This reaction mixture was then subjected to cycles of 95*C for 1 min, 50°C for 2 min and 72 0 C for 1.5 min. At the end of the cycling period, the reaction mixture was loaded on a 1% agarose gel and the material was electrophoresed for 2h after which the band at 1.3 kb was removed and the DNA recovered using the Gene Clean kit (Bio 101). This DNA was then digested with Ecoll, repurified and ligated to EcoRI digested pUC19 using T4 DNA ligase. The ligaticr mixture was used to transform competent E. coli DH5a. Recombinant plasmids were selected and sequenced. The insert was found to have a DNA sequence consistent with that of a class 2 porin. See, Murakami, K.

et aL, Infect. Immun. 57:2318-2323 (1989).

The plasmid pET-17b (Novagen) was used to express the class 2 porin. As described below, two plasmids were constructed that yielded two different proteins. One plasmid was designed to produce a mature class 2 porin while the other was designed to yield a class 2 porin fused to amino acids from the T7 gene 410 capsid protein.

WO 95/03413 W 0IV S94100327 Construction of the mature class 2 porin The mature class 2 porin was constructed by amplifying the pUC 19class 2 porin construct using the oligonucleotides (SEQ ID NO. 18) 5' CCT GTT GCA GCA CAT ATG GAC GTT ACC TTG TAC GOT ACA ATT AAA GC 3' and (SEQ ID NO. 19) 5 CGA CAG GCT TTT TCT CGA GAC CAA TCT TTT CAG This strategy allowed the cloning of the amplified class 2 porin into the Ndel and Xhol sites of the plasmid pET-17b thus producing a mature class 2 porin. Standard PCR was conducted using the pUC19-class 2 as the template and the two oligonucleotides described above. This PCR reaction yielded a 1.1kb product when analyzed on a agarose gel. The DNA obtained from the PCR reaction was gel purified and digested with the restriction enzymes Ndel and Xhol. The 1.lkb DNA produced was again gel purified and ligated to Ndel and Xhol digested pET-17b using T 4 DNA ligase. This ligation mixture was then used to transform competent E. 'coli DH5a. Colonies that contained the 1. lkb insert were chosen for further analysis. The DNA from the clones was analyzed by restriction mapping and the cloning junctions of the chosen plasmids were sequenced. After this analysis, the DNA obtained from the DH5a clones was used to transform E. coli BL21(DE3)-AompA.

The transformants were selected to LB-agar containing 100 ig/ml of carbenicillin. Several transformants were screened for their ability to make the class 2 perin protein. This was done by growing the clones in LB liquid medium containing 100 pg/ml of carbenicillin and 0.4% glucose at 0 C to ODo 0 0.6 then inducing the cultures with IPTG (0.4 mM). The cells were then disruptcd and the cell extract was analyzed by SDS-PAGE.

F -L 4 I WO 9$/03413 WO 93/03413IVII,9/t JO4IWIZ -36- Construction of Ilse fusion class 2,porln The fusion class 2 poenn wag Constructed by Wlli~fying tile pUG 19class 2 porin constn-iCt using the oligotitcleotides (SlVQ ID)NO. 20) 5' CCT OTT GA GO CAT GGA GAG OTT AGO TTG TAG GOT ACA ATT AAA GC 3' and (SEQ ID NO. 21) 5' MOA CG OCT TTT TCT OA GAG CAA TOT TTT CG This strategy allowed the cloning of the amplified class 2 pori into the IBatnfll and Viol sites of the plasniid pETl7b thus producing a fusion class 2 porin containing an additional 22 amino acids at the N-terminus derived from the T7 (b 10 calp(id protein contained in thle plasmid. Standard PCR was conducted using the pUG 19-class 2,as the template and the two oligonucleotides described ibove. The PCR reaction yielded a 1.1kb product when analyzed on a 1.0% agarose gel.

The DNA obtained from the PCR reaction was get purid and digestedl with the reaction enzymes BarnHik and Viol. The 1.,1kb product produced was again gel purified and ligated to BaiI and Viol digested pET'-17b using T 4 DNA ligase. This ligation mixture was then used to transform competent E. coi Ma.5x Colonics that contained the 1 .1kb insert were chosen for further analysis. The DNA from the DH15cx clones was analyzed by restriction enzyme mapping and the cloning junctions of the choseni plasmids were sequenced. After ths analysis, the DNA obtained from the clones was used to transform coi BL2l(DE3)-AonpA. The transformants were selected on LB-agar containing 100 l~g/rnl of carbenicitlin. Several transformants were screened for their ability to make the class 2 porin protein. This was done by growing the clones in LB liquid medium containing 100 iig/ml of carbeniciLlin and 0.4 glucose at to OD6. 0.6 then inducing the cultures with IPTO (0.4 mM). The cells were then disrupted and the cell extract was analyzed by SDS-PAGE.

WO 95/0.413 WO 95/3413 'MIUS940W.3Zi 37 Example 3. Cloning and Expression of the Mature class 3 p~orn front Group Ri Neisserla ineningilidis sirain 8765 in E. coil Genomic DNA was isolated from aipproximately 0,5 g of Group 'B Neisserla ineningitidis strain 8765 usinig the method described above (Siumbrook et al,, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring H~arbor, Now York, Cold Spring Marbor Labomato-y Press (1989)), This DNA dien served as the template for two class 3 porni spcciic oligonucleotides in a standard r-CR reaction, The mature class 3 porin was constructed by amplifying the genomnic DNA from 8765 using the oligonucleotides: (SfEQ ID NO. 22) 5' OTT OCA OCA CA7ATG GAG OTT ACC CTG TAC GO ACC 3' (SEQ ID NO. 23) and 5' GOG GOG ATO OAT CCA OAT TAO AAT TTG TG CG AGA CCG ACA CC This strategy allowed the cloning of the amplified class 3 porin into the NdeI and IBamI'I sites of the plasmid pBTthus producing a mature class 3 porin. Standard PCR was conducted using the genomic DNA isolated from 8765 as the template and the two oligonucleotides described above.

The reaction conditions were as folows: 8765 genomic DNA 200 ng, the two oligonucleotide primers described above at 1 1.tM of each, 200 IM of each dNTP, PCR reaction buffer (10 mM Tris H-Ii, 50 mM KCl, PH 1.5 mM MgCI 2 and 2.5 units of Taq polymerase, and made up to 100 with distilled watcy. This reaction mixture was then subjected to cycles of 95 0 C for 1 min, 50'C, for 2 min and 72'C for 13 Mhs PCR reaction yielded about 930 bp of product, as Wnaiyzed on a 1 agarose get. The DNA obtained from the PCR reaction was gel purified and digested with the restriction enzymes NdeI and Barn}JI. The 930 bp prodivct was again gel purified and ligated to Mdel and BamRl digested pET..24a(+I) using T4 ligase. This ligation mixture was then used to transform competent E. coli Mu~. Colonies that contained the 930 bp mnr~l~nra~Pnrx~manranmnansnm*nnnunr WO 95/03413 i/tJiS9408327 -38 insert were chosen for further analysis. The DNA from the E. coli clones was analyzed by restriction enzyme mapping and cloning junctions of the chosen plasmids were sequenced. After this analysis, the DNA obtained from the E. coli DH5a clones was used to transform E. coli BL21(DE3)-AompA. The transformants were selected on LD-agar containing 50 /g/ml of kanamycin. Several transformants were screened for their ability to make the class 3 porin protein. This was done by growing the clones in LB liquid medium containing 50 tg/ml of kanamycin and 0.4% cf glucose at 30*C to OD, 0.6 then inducing the cultures r with IPTG (1 mM). The cells were then disnrrted and the cell extract was analyzed by SDS-PAGE.

Example 4. Purification and refolding of recombinant class 2 porin E coli strain BL21(DE3)AompA [pNV-5] is grown to mid-log phase (OD 0.6 at 600 nm) in Luria broth at 30 0 C. IPTG is then added (0.4 mM final) and the cells grown an additional two hours at 37 0 C. The cells were then harvested and washed with several volumes of TEN buffer mM Tris-HC1, 0.2 M NaC1, 10 mM EDTA, pH 8.0) and the cell paste stored frozen at -75 °C.

For purification preweighed cells are thawed and suspended in TEN buffer at a 1:15 ratio The suspension is passed through a Stansted cell disrupter (Stansted fluid power Ltd.) twice at 8,000 psi, The resultant solution is then centrifuged at 13,000 rpm for 20 min and the supernatant discarded. The pellet is then twice suspended in TEN buffer containing deoxycbnlate and the supematants discarded. The pellet is then suspended in TEN buffer containing 8 M deionized urea (electrophoresis grade) and 0.1 mM PMSF (3 g/lOml). The suspension is sonicated for min or until an even suspension is achieved. 10 ml of a 10% aqueous 4 ~a~urnssnnai" ~qZqaCn~ R my WO 95/03413 W 54C1I'IUS94/i003Z7 39 solution of 3,14-zwittergen (Calbiochem) is added and the solution thoroughly mixed. The solution is again sonicated for 10 min. Any residual insoluble material is removed by centrifugation. The protein concentration is detetmined and the protein concentration adjusted to 2 mg/ml with 8 M urea-10% zwittergen buffer (1:1 ratio).

This mixture is then applied to a 2.6 x 100 cm column of Sephacryl S-300 equilibrated in 100 mM Tris-HC1, 1 M NaCI, 10 mM EDTA, mM CaC1 2 0.05% 3,14-zwittergen, 0.02% sodium azide, pH 8.0. The flow rate is maintained at 1 ml/min. Fractions of 10 ml are collected. The porin refolds into trimer during the gel filtration.. The OD 280 nm of each fraction is measured and those fractions containing protein are subjected to SDS gel electrophoresis assay for porin. Those fractions containing porin are pooled. The pooled fractions are either dialyzed or diluted 1:10 in 50 mM Tris HC1 pH 8.0, 0.05% 3,14-zwittergen, 5 mM EDTA, 0.1 M NaC1. The resulting solution is then applied to a 2.6 x cm Q sepharose high performance column (Pharmacia) equilibrated in the same buffer. The porin is eluted with a linear gradient of 0.1 to 1 M NaCI.

Example 5. Purification and refolding of recombinant class 3 porin E coli strain BL21 (DE3) AompA containing the porB-pET-17b plasmid is grown to mid-log phase (OD 0.6 at 600 nm) in Luria broth at 30 C. IPTG is then added (0.4 mM final) and the cells grown an additional two hours at 37 0 C. The cells were then harvested and washed with several volumes of TEN buffer (50 mM Tris-HC1, 0.2 M NaCI, mM EDTA, pH 8.0) and the cell paste stored frozen at -75 C.

For purification about 3 grams of cells are thawed and suspended in 9 ml of TEN buffer. Lysozyme is added (Sigma, 0.25 mg/ml) r C-Il I~ ~I~OLI- .l r WO 95/03413 W 90 3VCV/IU8432 40 deoxycholate (Sigma, 1.3 mg/ml) plus PMSF (Sigma, C/g/ml) and the mixture gently shaken for one hour at room temperature. During this time, the cells lyse and the released DNA causes the solution to become very viscous. DNase is then added (Sigma, 2 /g/ml) and the solution again mixed for one hour at room temperature. The mixture is then centrifuged at 15K rpm in a S-600 rotor for 30 minutes and the supernatant discarded.

The pellet is then twice suspended in 10 ml of TEN buffer and the supernatants discarded. The pellet is then suspended in 10 ml of 8 M urea (Pierce) in TEN buffer. The mixture is gently stirred to break up any clumps. The suspension is sonicated for 20 minutes or until an even suspension is achieved. 10 ml of a 10% aqueous solution of 3,14zwittergen (Calbiochem) is added and the solution thoroughly mixed. The solution is again sonicated for 10 minutes. Any residual insoluble material is removed by centrifugation. The protein concentration is determined and the protein concentration adjusted to 2 mg/ml with 8 M zwittergen buffer (1:1 ratio).

This mixture is then applied to a 180 x 2.5 cm column of Sephacryl S-300 (Pharmacia) equilibrated in 100 mM Tris-HCI, 1 M NaCI, 10 mM EDTA, 20 mM CaCl 2 0.05% 3,14-zwittergen, pH 8.0. The flow rate is maintained at 1 ml/min. Fractions of 10 ml are collected. The porin refolds into trimer during the gel filtration. The OD 28 o nm of each fraction is measured and those fractions containing protein are subjected to SDS gel electrophoresis assay for porin. Those fractions containing porin are pooled.

The pooled fractions are dialyzed and concentrated 4-6 fold using Amicon concentrator with a PM 10 membrane against buffer containing 100 mM Tris-HC1, 0.1 M NaC1, 10 mM EDTA, 0.05% 3,14-zwittergen, pH 8.0. Alternativeity, the pooled fractions are precipitated with ethanol and resuspended with the above-mentioned buffer. Six to 10 mg -rr~na~l WO 95/03413ll W 1CT[S94108327' -41 of the material is then applied to a monoQ 10/10 column (Pharmacia) equilibrated in the same buffer. The porin is eluted from a shallow 0.1 to 0.6 M NaCI gradient with a 1.2% increase per min over a 50 min period.

The Flow rate is 1 ml/min. The peak containing porin is collected and dialyzed against TEN buffer and 0.05% 3,14-zwittergen. The porin may be purified further by another S-300 chromatography.

Example 6. Purification and chemical modification of the polysaccharides The capsular polysaccharide from both group B Neisseria meningitidis and E. coli K1 consists of ao(2-8) polysialic acid (commonly referred to as GBMP or K1 polysaccharide). High molecular weight polysaccharide isolated from growth medium by precipitation (see, Frasch, "Production and Control of Neisseria meningitidis Vaccines" in Bacterial Vaccines, Alan R. Liss, Inc., pages 123-145 (1990)) was purified and chemically modified before being coupled to the porin protein. The high molecular weight polysaccharide was partially depolymerized with 0.1 M acetic acid (7 mg polysaccharide/ml), pH 6.0 to 6.5 (70 0 C, 3 hrs) to provide polysaccharide having an average molecular weight of 12,000- 16,000. After purification by gel filtration column chromatography (Superdex 200 prep grade, Pharmacia), the polysaccharide was N-deacetylated in the presence of NaBH4 and then N-propionylated as described by Jennings et al. Immunol. 137:1808 (1986)) to afford N-Pr GBMP. Treatment with NaIO 4 followed by gel filtration column purification gave the oxidized N-Pr GBMP having an average molecular weight of 12,000 daltons.

I b r r ~L-9- WR~Z* ~ZIIIBP~lrr*~SRCi-I-+i~7i~~l ~II WO 95/03413 WO 95/3413 Tf(94/Io37 -42- Example 7. Coupling of oxidized N-Pr GBMP to the group B meningococcal class 3 porin protein (PP) The oxidized N-Pr GBMP (9.5 mg) was added to purified class 3 porin protein (3.4 mg) dissolved in 0.21 ml of 0.2 M phosphate buffer pH 7.5 which also contained 10% octyl glucoside. After the polysaccharide was dissolved, sodium cyanoborohydride (7 mg) was added and the reaction solution was incubated at 37°C for 4 days. The reaction mixture was diluted with 0.15 M sodium chloride solution containing 0.01% thimerosMa and separated by gel filtration column chromatography using Superdex 200 PG. The conjugate (N-Pr GBMP-PP) was obtained as single peak eluting near the void volume. Analysis of the conjugate solution for sialic acid and protein showed that the conjugate consists of 43% polysaccharide by weight. The porin protein was recovered in the conjugate in 44 yield and the polysaccharide in 12% yield. The protein recoveries in different experiments generally occur in the 50-80% range and those of the polysaccharide in the 9-13% range.

Example 8. Immunogenicity studies The immunogenicities of the N-Pr GBMP-PP conjugate and those of the N-Pr GBMP-Tetanus toxoid (N-Pr GBMP-TT) conjugate which was prepared by a similar coupling procedure were assayed in 4-6 week old outbread Swiss Webster CFW female mice. The polysaccharide (2 Ag)conjugate was administered on days 1, 14 and 28, and the sera collected on day 38. The conjugates were administered as saline solutions, adsorbed on aluminum hydroxide, or admixed with stearyl tyrosine. The sera ELISA titers against the polysaccharide antigen and bactericidal titers against N.

meningitidis group B are summarized in Table 1.

WO 95/03413 PCT/US94/08327 43 Having now fully described this invention, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations and other perimeters without affecting the scope of the invention or any embodiment thereof.

All patents and publications cited herein are fully incorporated by reference herein in their entirety.

rr- -_st q g M I -1 WO 95/03413 PCT/US94/08l32 -44- Table 1.

ELISA and Bactericidal Titers of Group B Meningococcal Conjugate Vaccines (N-Pr GBMP-Protein) Bactericidal Vaccine Adjuvant ELISA Titer Titer N-Pr GBMP-TT Saline 5,400 0

AI(OH)

3 13,000 0 ST' 17,000 0

CFA

2 40,000 800 N-Pr GBMP-PP Saline 20,000 500 Saline 22,000 150 Saline 39,000 960

AI(OH)

3 93,000 200 A1(OH) 3 166,000 3,200 Al(OH) 3 130,000 1,200 ST 53,000 1,000 ST 29,000 1,700 ST 72,000 1,500 N-Pr GBMP Saline 100 0 Al(OH) 3 100 0 ST 100 0 PP Saline 100 0

AI(OH)

3 100 0 ST 660 0 1 ST Stearyl tyrosine.

2 CFA Complete Freund's Adjuvant s I I WO 95/03413 WPCTJAI94/0831s SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: The Rockefeller University 1230 York Avenue New York, New York 10021 United States of America North American Vaccine, Inc.

12103 Indian Creek Court Beltsville, Maryland 20705 United States of America INVENTORS: Blake, Milan B.

Tai, Joseph Y.

Qi, Huilin L.

Liang, Shu-Mei Hronowski, Lucjan J.J.

Pullen, Jeffrey K.

(ii) TITLE OF INVENTION: Method for the High Level Exrression, Purification and Refolding of the Outer Membrane Group B Porin Proteins from Neisseria Meningitidis (iii) NUMBER OF SEQUENCES: 23 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Sterne, Kessler, Goldstein Fox STREET: 1100 New York Ave., Suite 600 CITY: Washington STATE: D.C.

COUNTRY: USA ZIP: 20005-3934 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: To be Assigned FILING DATE: Herewith

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: Application No.: US 08/096,182 Filing Date: 23 July 1993 (viii) ATTORNEY/AGENT INFORMATION: NAME: Esmond, Robert W.

REGISTRATION NUMBER: 32,893 REFERENCE/DOCKET NUMBER: 1433.006PC00 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (202) 371-2600 TELEFAX: (202) 371-2540 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 930 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: both (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..930 WO 95/O3413 P('T/(JS94/O83 Z7 (xi) SEQUENCE DESCRXPTION; SEQ ID NO-1: TTG TAC GGT ACA ATT Leu Tyr Gly Thr 1 CAC CAG AAC GGC His Gin Asn Gly GAT TTG GGT TCG Asp Leu Gly Ser GGC CTG AAA GCC Gly Leu Lys Ala ACT GAC TCC GGT Thr Asp Ser Gly GGC TTC GGT AAA Gly Phe Gly Lys ACC GGC GAC ATC Thr Giy Asp Ile 100 AAC AAA ATT GCC Asn Lys Ile Ala 115 TCT CCC GAA TTT Ser Pro Giu Phe 130 GAC AAT GCA GGC Asp Asn Ala Gly 145 TAC AAA AAC GGT TFyr Lys Asn Gly CAT CAT CAA GTG I His His Gin Va.

180 CGT TTG GTC AGC Arg Leu Val Ser 195 GTA CAG CAA CAA Val Gin Gin Gin 210 TCT CM ACC GAA Ser Gin Thr Glu 225 ACG CCC CGA GTT '2 Thr Pro Arg Vai GCA GAC ATA GGC I Ala Asp Ile Giy I 260 Ile 5

CAA

Gin

AAA

Lys

ATT

Ile

TGG

Trp

TTG

Len

AAT

Asn

GAA

Glu

GCC

Ala

AGA

Arg

GGC

Gly 165

CAA

3Gl

GGT

3iy 3AC .sp 3TT Val

['CT

3er

!AC

ksn AAA GCA Lys Ala OTT ACT Val Thr ATC GGC Ile Gly TGG CAG Trp Gin 55 GGC AAC Gly Asn 70 CGC GTC Arg Val CCT TGG Pro Trp CCC GAG Pro Glu GGC CTC Gly Len 135 CAT AAC.

His Asn 150 TTC TTC Phe Phe GAG GGC Glu Gly GMA ACT TCC CGC Glu Thr Ser Arg 10 ACA ACC GCT ACC Thr Thr Ala Thr GGC CAA GAA GAC Gly Gin Glu Asp CAA AAA GCA TCT Gin Lys Ala Ser TCC TTC ATC GGC Ser Phe lie Gly 75 TTG AAC AGC GTC Len Asn Ser Val TCT GTA TTT Ser Val Phe GGC ATC GTT Gly le Val CTC GGT AAC Leu Gly Asn ATC GCC GGT Ile Ala Gly CGC CAA Arg Gin GGT CGT Gly Arg GAT AGC Asp Ser 105 GCA CGC Ala Arg 120 AGC GGC Ser Gly AGC GAA Ser Glu GTG CAA Val Gin TTG AAT Leu Asn 185 AT GAT %sn Asp 200 CTG ACT Aeu Thr kCC TTG Lhr Len AC GGC lis Gly TTG AAA Len Lys

CTG

Leu 90

AAA

Lys

CTC

Len

AGC

Ser

TCT

Ser

TAT

Tyr 170

ATT

Ile

GCC

Ala

GAT

Asp.

GCA

Ala

TTC

Phe 250

GTG

Val 1

AGC

Ser

ATT

Ile

GTA

Val

TAC

Tyr 155

GGC

Gly

GAG

Glu

CTG

Leu

GCT

Ala

TAC

Tyr 235

AA

Lys

OTT

Va1

GAC

Asp

TCC

Ser

CAA

Gin 140

CAC

His

GOT

Gly

AAA

Lys

TAC

Tyr

TCC

Ser 220

CGC

Arg

GOT

Gly

GTC

Val

TAT

Tyr

GTA

Val 125

TAC

Tyr

GCC

Ala

GCC

Ala

TAC

Tyr

GCT

Ala 205

AAT

Asn

TTC

Phe

TTG

Leu

GGT

Gly

TTG

Len 110

CGC

Arg

GCG

Ala

GGC

Gly

TAT

Tyr

CAG

Gln 190

TCC

Ser

TCG

Ser

GGC

Gly

GTT

Val

GCG

Ala 270

AAA

Lys

GOT

Gly

TAC

Tyr

CTT

Len

TTC

Phe

AAA

Lys 175

ATT

Ile

GTA

Val

CAC

His

AAC

Asn

GAT

Asp 255

GAA

Glu

GGC

Gly

GAC

Asp

GTA

Val

GAT

Asp

AAC

Asn

AAC

Asn 160

AGA

Arg

CAC

His

GCC

Ala

AAC

Asn

GTA

Val 240

GAT

Asp

TAC

Tyr 48 96 144 192 240 288 336 384 432 480 528 576 624 672 720 768 816 c c

GAC

Asp

AAA

Lys 215

GCT

Ala 0CC Ala 2

I

C

I

TAC GAC CAA Tyr Asp Gin 265 IIL~_ -I P I .WO 95/03413 V CT( 01804 /0 8127

GAC

Asp

GAA

Gin

CTG

Leu 305 TTC TCC MAA CGC Phe Ser Lys Arg 27S GGC AAA GGC GMA Gly Lys Gly Gin 290 CGT CAC AAA TTC Arg His Lys Phe

ACT

Thr

AAC

Asn

TMA

310 TCT GCC TTO OTT TCT OCC GGT TOG TTG CMA Ser Ala Lou Val Szr Ala Oly Trp LOU Gln 280 285 AAA TTC GTA GCG ACT GCC GGC GGT GTT GOT Lys Phe Val Ala Thr Ala Gly Gly Val Gly 295 300 INFORMATION FOR SEQ ID NO:2; Wi SEQUENCE CHARACTERISTICS: LENGTH: 309 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE Lou Tyr Gly Thr Ile 1 5 His Gin Asn Gly Gin Asp Leu Gly Ser Lys Gly Leu Lys Ala Ile Thr Asp Ser Gly Trp Gly Phe Gly Lys Len Thr Gly Asp Ile Asn 100 Asn Lys Ile Ala Gin 115 Ser Pro Giu Phe Ala 130 Asp Asn Ala Gly Arg 145 Tyr Lys Asn Gly Gly 165 His His Gin Vai Gin 180 Arg Leu Val Ser Gly 195 DESCRIPTION: SEQ ID NO:2: Lys Ala Gly Val Gin Thr Ser Arg Ser Val. Phe 10 Val Ile Trp, Gly 70 Arg Pro Pro Gly His 150 Phe Gin Tyr Thr Gly Gin 55 Asn Val TXp Giu Leu 135 Asn Phe Gly Asp Giu Phe 40 Val Arg Gly Asp Ala 120 Ser Ser Val Leu Asn 200 Thr Thr Ala Gly Gin Gin Gin Lys Ala Ser Phe Ile Len Asn Ser 90 Lys Ser Asp Len Ile Ser Ser Val Gin 140 Ser Tyr His 155 Tyr Gly Gly 170 Ile Gin Lys Ala Len Tyr Thr Giy Ile Asp Leu Gly Ser Ile Ala Gly Len Lys Val Leu Lys Tyr Len Gly 110 Vai Arg Tyr 125 Tyr Ala Len Ala Gly Phe Ala Tyr Lys 175 Tyr Gin Ile 190 Ala Ser Val 205 Val Gin Gin Gin Asp Ala 210 Lys Len Thr Asp Ala Ser 215 220 Asn Ser His Asn Wo OND/04 .10MUS-94/08327 Ser 225 Thr Ala Asp Glu Leu 305 Ala Ala Thr Lou Ala Tyr Arg Phe Gly Asn Val 230 235 240 Tyr' Ala His Gly Phe Lys Gly Lou Val Asp Asp 250 255 Glu Tyr Asp Gin Val. Val Val. Gly Ala Giu Tyr 265 270 Thr Ser Ala Lou Val Ser Ala Gly Trp Lou Gin 280 285 Asn Lys Phe Val Ala Thr Ala Gly Gly Val Gly 295 300 INFORMATION FOR SEQ ID NO:3: Wi SEQUENCE CHARACTERISTICSi LENGTH: 1029 base pairs TYPE: nucleic acid STRANDEDNESS. double TOPOLOGY: both (ix) FEATURE: NAME/KEY: CDS LOCATION: 1.-1029 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATG GAC GTT ACC TTG TAC GGT ACA ATT AAA GCA GGC GTA GAA GTT TCT Met Asp Val Thr Leu Tyr 1

CGC

Arg

ACT

Thr

CAA

Gin

AAA

Lys

TTC

Phe

AAC

Asn

GGT

Gly

AAA

Lys

ACC

Thr 35

GAC

Asp

TCC

Ser

GGC

Gly

GTA

Val

AAC

Asn 115

GAT

Asp 20

CAA

Gin

CTC

Lou

ATC

Ile

TTG

Leu

TTG

Lou 100

ACC

Thr 5 GCT GGT Ala Gly ATT GCC Ile Ala GGC AAC Gly Asn GCC GGC Ala Gly 70 AAA GGC Lys Gly AAA GAC Lys Asp GAA GAT Giu Asp Gly Thr Ile Lys .10 ACA TAT AAA GCT Thr Tyr Lys Ala 25 GAC TTC GGT TCT Asp Phe Gly Ser 40 GGC ATG AAA GCC Gly Met Lys Ala 55 ACT AAC AGC GGC Thr Asn Ser Gly GGC TTC GGT ACC Gly Phe Gly Thr 90 AGC GGC GAC AAC Ser Gly Asp Asn 105 GTA CTG GGA CTG Val Leu Gly Lou 120 Ala Gly Val Glu Va. Ser

CAA

Gln

AAA

Lys

ATT

Ile

TGG

Trp 75

GTC

Val

GTC

Val

GGT

Gly GGC GGA Gly Gly ATC GGT Ile Gly 45 TGG CAG Trp Gin 60 GGT AAC Gly Asn CGC GCC Arg Ala AAT GCA Asfl Ala ACT ATC Thr Ile 125

AAA

Lys 30

TTC

Phe

TTG

Leu

CGC

Arg

GGT

Gly

TGG

Trp 110

GGT

Gly

TCT

Ser

AAA

Lys

GAA

Glu

CAG

Gin

AAT

Asn

GAA

Glu

CGT

Arg 48 96 144 192 240 288 336 384 .WO 95/03413 .110YU894/00327 GAA ACC Glu Ser 130 COT GMA ATC TCC Arg Glu Ile Sexr

OTA,

Va~l 1 CGC 'VAC GAC TCT Arg Tyr Asp Ser GTA TTT OCA GGC Val Phe Al.a Gly

TTO

Phe 145 AGC GGC AGC GTA Ser Gly Ser Val TAC GTT CCG COO Tyr Val Pro Arg AAT GCG AAT GAT Asn Ala Asn Asp GAT AAM TAC AMA Asp Lys Tyr Lys

CAT

His 165 ACG R~AG TCC AGC Thr Lys Ser Ser GAG TOT TAO CAC Glu Ser Tyr His 000 GOT Ala Gly 175 OrG AMA TAO Leu Lye Tyr 000 AMA TAT Ala Lys Tyr 195 AAT GOC GOT TTO Asn Ala Gly Phe GOT CAA TAO OCA Gly Gin Tyr Ala GOT TOT TTT Oly Ser Phe 190 GOA OTA MAT Ala Val Asn OT GAT TTG MOC Ala Asp Leu Asn OAT GOA GM COT Asp Ala Olu Arg ACT OOA MAT 000 CAT OCT Thr Ala Asn Ala His Pro 210

OTT

Val 215 MOG OAT TAO CAA Lys Asp Tyr Gin CAC 000 OTA OTT His Arg Val Val 000 GOT TAO OAT 000 MAT GAO OTO TACOGTT TOT OTT 000 OT' OAO TAT Ala Oly Tyr Asp Ala Asn Asp Leu Tyr Val Ser Val Ala Gly Gln Tyr 225 230 235 240 GM OT OCT AMA 01u Ala Ala Lys

MOC

Asn 245 MOC GAO OTT GOT Aen Glu Val Gly ACC MOG GOT AAA Thr Lys Gly Lye AA CAC Lys His 255 GAG CM.A ACT Olu 0Th Thr AOO OOT 000 Thr Pro Arg 275

CAA

Oln 260 OTT 000 OT ACT Val Ala Ala Thr OT TAO CGT TTT Ala Tyr Arg Phe 000 MOC OTA Oly Asn Val 270 OTO MAT 000 Val Aen Gly OTT TOT TAO 000 Val Ser Tyr Ala 000 TTO AMA OCT Gly Phe Lye Ala

AMA

Lys 285 OTO AMA GAO GOA MAT TAO CMA TAO GAO CAA OTT ATO Val Lys Asp Ala Aen Tyr Oln Tyr Asp Gin Val Ile 290 295 300 OTT GOT 000 GAO Val Oly Ala Asp

TAO

Tyr 305 GAO TTO TOO AAA Asp Phe Ser Lys 000 Arg 310 ACT TOO OT OTO OTT TOT 000 GOT TOG Thr Ser Ala Leu Val Ser Ala Gly Trp 315 AMA CMA GOT AMA Lys Gin Gly Lye 000 000 Gly Ala 325 OGA AMA OTC Gly Lye Val CMA ACT 000 AGO Oln Thr Ala Ser ATO OTT Met Val 335 1008 1029 GOT OTO COT Gly Leu Arg AMA TTO TMA Lys Phe INFORM4ATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 342 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 9$/03413 PKi"VfUS94/00,732 (xi) SEQUENCO DEOCRTXONt 30I 10 N:4i Met Asp Val Thr

I

Arg Thr Gin Lye Phe Asn Gly Glu Phe 145 Asp Leu Ala Thr Ala 225 Glu Glu Thr Val Tyr 305 Lys Gly Val Lye Ala Thr Glu Asp Ala Ser Ile Gly Thr Val Ser Asn 115 Ser Arg 130 Ser Gly Lye Tyr Lye Tyr Lye Tyr 195 Ala Asn 210 Gly Tyr Ala Ala Gin Thr Pro Arg 275 Lye Asp 290 Asp Phe Gin Gly Leu Arg i Asp Gin Leu Ile Leu Leu 100 Thr Glu Ser Lye Glu 180 Ala Ala Asp Lye Gin 260 Val Ala Ser Lys His 340 Leu 5 Ala Ile Gly Ala Lye Lye Giu Ile Val His 165 Asn Asp His Ala Asn 245 Val Ser Asn Lye Gly 2 325 Lye I Tyr Gly Thr Ile Lys Ala Gly Val Glu Val $er 10 is Gly Ala Asn Gly 70 Gly Asp Asp Ser Gin 150 Thr Ala Leu Pro Asn 230 Asn kla ryr ryr krg 10 Thr Asp Gly 55 Thr Gly Ser Va.

Val 135 Tyr Lye Gly Asn Val 215 Asp Glu Ala Ala Gin 295 Thr Tyr Lys 25 Phe Gly 40 Met Lye Asn Ser Phe Gly Gly Asp 105 Leu Gly 120 Arg Tyr Val Pro Ser Ser Phe Phe 185 Thr Asp 200 Lye Asp Leu Tyr Val Gly Thr Ala 265 His Gly 280 Tyr Asp Ser Ala Ala Gin Gly Ser Lys Ile Ala Ile Trp Gly Trp Gly 75 Thr Val Arg 90 Asn Val Asn Leu Gly Thr Asp Ser Pro 140 Arg Asp Asn 155 Arg Giu Ser 170 Gly Gin Tyr Ala Giu Arg Tyr Gin Val 220 Val Ser Val 235 Ser Thr Lye 250 Ala Tyr Arg Phe Lye Ala 2 Gin Val Ile 300 Leu Val Ser Gly Gly Gin Asn Ala Ala Ile 125 Val Ala Tyr Ala Val 205 His Ala Gly Phe Lys 1 285 Lye Phe Leu Arg Gly Trp 110 Gly Phe Asn His Gly 190 Ala Arg Gly Lye Gly 270 Vtal Ser Lye Glu Gin Asn Glu Arg Ala Asp Ala 175 Ser Val Val Gin Lye I 255 Asn 1 Asn Lys Gly Gin Ser Leu Ser Val Gly Val 160 Gly Phe Asn Val Tyr 240 His Val Gly V7al Gly Ala Asp kla Gly 315 Gin Thr Ala Ser Trp Leu 320 Met Val 335 ~la Gly Lye Val Glu 330 ?he I-Y- *WO 95/03413 S1 INFORMATION F'OR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1092 base pairs TYPE, nucleic acid STRANDEDNESS: double TOPOLOGY, both (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .1092 (xi) SEQUENCE DESCRIPTION: SEQ ID ATG OCT AGC ATG ACT GOT OGA CAG CAA ATG OGT Met. Ala Ser Met Thr Gly Gly Gin Gin Met Oly PCTA3894/00327 COO GAT TCA AGC TTG Arg Asp Ser Ser Leu

I

GTA

Val1

GGC

Gly

GGC

Gly

ATC

Ile

TGG

Trp GGT AAC CGC Gly Asn Arg CGC 0CC GOT Arg Ala Oly 115 AAT GCA TGG Asn Ala Trp 130 ACT ATC GOT Thr Ile Gly 145 CCC GTA TTT Pro Val Phe AAT OCO AAT Asn Ala Asn TCT TAC CAC Ser Tyr His 195

TCG

Ser 20

OTT

Val1

TCT

Ser

AAA

Lys

GAA

Glu

CAG

Gin 100

AAT

Asn

GAA

Glu

CGT

Arg

GCA

Ala

GAT

Asp 180

GCC

Ale~ GAT CCA GAC OTT Asp Pro Asp Val TCT COC GTA ZAA Ser Arg Val Lys AAA ACT GCA ACC Lys Thr Ala Thr 55 GOT CAA GAA GAC Oly Gin Glu Asp 70 CAA AAA GCC TCC Gin Lys Ala Ser TCC TTC ATC GOC Ser Phe Ile Oly CTG AAC ACC OTA Leu Asri Thr Val 120 TCT OGT TCT AAC Ser Gly Ser Asn 135 GTA GAA AGC CGT Val Gin Ser Arg 150 GOC TTC AOC 000 Oly Phe Ser Gly 165 GTO GAT AAA TAC Val Asp Lys Tyr GOT CTG AAA TAC Gly Leu Lys Tyr 200

ACC

Thr

OAT

Asp

CAA

Gin

CTC

Leu

ATC

Ile

TTG

Leu 105

TTG

Leu

ACC

Thr

GAA

0Th.

AGC

Ser

~AA

Lys 185

GAA~

Glu

GGC

Oly

GGC

Gly

GAC

Asp

OAT

Asp

TCC

Ser 155

CAA

Gin

ACG

Thr 0CC Ala TTG TAC Leu Tyr GCT GOT Ala Oly ATT 0CC Ile Ala GGC AAC Oly Asn

GOT

Gly

ACA

Thr

GAC

Asp

GGC

Oly

ACT

Thr

GC

Gly

AGC

Ser

OTA

Val 140

GTA

Val

TAC

Tyr

AAG

Lys

GGT

Gly AAA GCA Lys Ala OCT CAA Ala Gin TCT AMA Ser Lys AAC AOC Asn Ser TTC GOT Phe Oly 110 GOC GAC Gly Asp 125 CTG OGA Leu Gly COC TAO Arg Tyr GTT CO Val Pro

GGC

Gly

ACC

Thr

AAC

Asn

CTG

Leu

GAC

Asp

COC

Arg 175 ATO AAA GCC Met Lys Ala

TOT

Ser 160

OAT

Asp 1'CC AGC CGT GAG Ser Ser Arg Oiu 190 TTC TTC OGT CAA Phe Phe Gly Gin WO 95/03413 PeTiUS94/08321 52 TAC GCA GGT TCT TTT GCC AAA TAT GCT GAT TTG AAC ACT GAT GCA GAA 672 Tyr Ala Gly Ser Phe Ala Lys Tyr Ala Asp Leu Aen Thr Asp Ala Glu 210 215 220

CGT

Arg 225

GTA

Val

GTT

Val

AAG

Lys

CGT

Arg

GCT

Ala 305

ATC

Ile

TCT

Ser GTT GCA GTA Val Ala Val AAT ACT GCA AAT UCC Asn Thr Ala Asn Ala 230 GTA GTT GCC Val Val Ala 245 CAG TAT GAA Gin Tyr Glu 260 AAA C C GAG Lys g Glu AAC GTA ACG Asn Val Thr AAT GGC GTG Asn Gly Val 310 GCC GAC TAC Ala Asp Tyr 325 TGG TTG AAA Trp Leu Lys 340 GGT TAC GAT Gly Tyr Asp GCT GCT AAA Ala Ala Lys 265 CAA ACT CAA Gin Thr Gin 280 CCT CGC GTT Pro Arg Val 295 AAA GAC GCA Lys Asp Ala GAC TTC TCC Asp Phe Ser CAA GGT AAA Gln Gly Lys 345

GTT

Val

GAC

Asp

CAG

Glu

GCT

Ala

GCC

Ala 300

CAA

Gin

ACT

Thr

GGA

Gly AAG GAT Lys Asp CTG TAC Leu Tyr GTT GGT Val Gly 270 ACT GCC Thr Ala 485 CAC GGC His Gly TAC GAC Tyr Asp TCC GCT Ser Ala AAA GTC Lys Val 350 TAC CAA Tyr Gin 240 GTT TCT Val Ser 255 TCT ACC Ser Thr GCT TAC Ala Tyr TTC AAA Phe Lys CAA GTT Gin Val 320 CTG GTT Leu Val 335 GAA CAA Glu Gin 720 768 816 864 912 960 1008 1056 1092 ACT GCC AGC Thr Ala Ser 355 ATG GT GGT CTG Met Val Gly Leu CAC AAA TTC TAA His Lys Phe INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 363 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met A3a Ser Met Thr Gly Gly Gin Gin Met Gly Arg Asp Ser Ser Leu 1 5 10 Val Pro Ser Ser Asp Pro Asp Val Thr Leu Tyr Gly Thr Ile Lys Ala 25 Gly Val Glu Val Ser Arg Val Lys Asp Ala Gly Thr Tyr Lys Ala Gin 40 Gly Gly Lys Ser Lys Thr Ala Thr Gin Ile Ala Asp Phe Gly Ser Lys 55 Ile Gly Phe Lys Gly Gin Glu Asp Leu Gly Asn Gly Met Lys Ala Ile 70 75 WO 95/03413 PCTIUS94/08327 53 Trp Gln Leu GIu Gln Lyo Ala Ser 1 I Ala Gly Thr Ann Set Gly Trp 90 Gly Arg Asn Thr 145 Pro Asn Ser Tyr Arg 225 Val Val Lys Arg Ala 305 Ile Ser Thr ASn Ala Ala 130 Ile Val Ala Tyr Ala 210 Val His Ala Gly Phe 290 Lys Val Ala Ale Arg Gln Ser 100 Gly Asn Leu 115 Trp Glu Ser Gly Arg Val Phe Ala Gly 165 Aan Asp Val 180 His Ala Gly 195 Gly Ser Phe Ala Val Aan Arg Val Val 245 Gly Gln Tyr 260 Lys Lys His 275 Gly Asn Val Val Asn Gly Gly Ala Asp 325 Gly Trp Leu 340 Ser Met Val 355 Phe Asn Gly Glu 150 Phe Asp Leu Ala Thr 230 Ala Glu Glu Thr Val 310 Tyr Lys Gly Ile Gly Thr Val 120 Ser Asn 135 Ser Arg Ser Gly Lys Tyr Lys Tyr 200 Lys Tyr 215 Ala Ann Gly Tyr Ala Ala Gin Thr 280 Pro Arg 295 Lys Asp Asp Phe Gin Gly Leu Arg 360 Leu 105 Leu Thr Glu Ser Lys 185 Glu Ala Ala Asp Lys 265 Gin Val Ala Ser Lys 345 His Lys Lys

GIU

Ile Val 170 His Asn Asp His Ala 250 Asn Val Ser Asn Lys A 330 Gly A Lys P Gly Giy Phe Gly Thr Val 110 Asp Ser Gly Asp Ann Val 125 Asp Val Leu Gly Leu Gly 140 Ser Val Arg Tyr Asp Ser 155 160 Gln Tyr Val Pro Arg Asp 175 Thr Lys Ser Ser Arg Glu 190 Ala Gly Phe Phe Gly Gin 205 Leu Asn Thr Asp Ala Glu 220 Pro Val Lys Asp Tyr Gin 235 240 Asn Asp Leu Tyr Val Ser 255 Asn Glu Val Gly Ser Thr 270 Ala Ala Thr Ala Ala Tyr 285 'yr Ala His Gly Phe Lys 300 Tyr Gln Tyr Asp Gin Val 15 320 Arg Thr Ser Ala Leu Val 335 la Gly Lys Val Glu Gin 350 'he INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 187 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: both (ix) FEATURE: NAME/KEY: CDS LOCATION: 101..187 p-- WO 95/03413 iCTI/U94/08327 54 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG AGACCACAAC GGTTTCCCTC TAGAAATAAT TTTGTTTAC TTAAAGAAGG AGATATACAT ATG GCT AGC ATG ACT 115 Met Ala Ser Met Thr 1 GGT GGA CAG CAA ATG GGT CGG GAT TCA AGC TTG GTA CCG AGC TCG GAT 163 Gly Gly Gin Gin Met Gly Arg Asp Ser Ser Leu Val Pro Ser Ser Asp 15 CTG CAG GTT ACC TTG TAC GGT ACA 187 Leu Gin Val Thr Leu Tyr Gly Thr INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 29 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ala Ser Met Thr Gly Gly Gln Gln Met 1 5 10 Val Pro Ser Ser Asp Leu Gin Val Thr Leu NO:8: Gly Arg Asp Ser Ser Leu Tyr Gly Thr INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 54 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: both (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..24 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GTT GGT CTG CGT CAC AAA TTC TAACTCGAGC AGATCCGGCT GCTAACAAAG Val Gly Leu Arg His Lys Phe 1

CCC

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Val Gly Leu Arg His Lys Phe 1 L~ -s SWO 95/03413 I'CTIUS94/08327 INFORMATION FOR SEQ ID NOill: SEQUENCE CHARACTERISTICS: LENGTH: 47 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GGGGTAGATC TGCAGGTTAC CTTGTACGGT ACAATTAAAG CAGGCGT 47 INFORMATION FOR SEQ ID NO;12: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS; single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION; SEQ ID NO:12: GGGGGGGTGA CCCTCGAGTT AGAATTTGTG ACGCAGACCA AC 42 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TCAAGCTTGG TACCGAGCTC INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY; linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: TTTGTTAGCA GCCGGATCTG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID CTCAAGACCC GTTTAGAGGC C 21 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear

I

WO 95/03413 PCT/US94/08327 56 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: AGCGGCTTGG AATTCCCGGC TGGCTTAAAT TTC 33 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CAAACGAATG AATTCAAATA AAAAAGCCTG INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 47 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CCTGTTGCAG CACATATGGA CGTTACCTTG TACGGTACAA TTAAAGC 47 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: CGACAGGCTT TTTCTCGAGA CCAATCTTTT CAG 33 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 47 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID CCTGTTGCAG CGGATCCAGA CGTTACCTTG TACGGTACAA TTAAAGC 47 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CGACAGGCTT TTTCTCGAGA CCAATCTTTT CAG 33 INFORMATION FOR SEQ ID NO:22: *WO 9S/03413 P'fS)/8z MS1NCS CWAACWVRI8TXCS; LENGTH-. 36 base pairs TYPE: nucleic acid STRANDEDNESS, single TOPOLOGY; linear (xi) SEQUENCE DESCRIPTXONt SEQ ID NO:22: GTTGCAGCAC ATATGGACGT TACCCTGTAC GGCACC 3 INFORMATION FOR SEQ ID NO:23; SEQUENCE CHARACTERISTICS: LENGTH: 44 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GGGGGGATGG ATCCAGATTA GAATTTGTGG CGCAGACCGA CACC 44

Claims (16)

1. A method for the high level expression of the outer membrane meningococcal group B porin protein or fusion protein thereof in an E. coli host cell having a deleted ompA gene (AompA), comprising: transforming the E. coli AompA host cell with a vector comprising a selectable marker and a gene coding for a protein selected from the group consisting of: a mature porin protein, and (ii) a fusion protein which is a mature porin protein fused to amino acids 1 to or 1 to 22 of the T7 gene 410 capsid protein; wherein said gene is operably linked to the T7 promoter; growing the transformed host cell in a culture medium containing a selection agent, and inducing expression of said protein; wherein the protein so expressed comprises more than about 2% of the total protein expressed in the host cell.

2. The method according to claim 1, wherein said prGtein is the mature group B class 2 porin protein.

3. The method according to claim 1, wherein said protein is the mature group B class 3 porin protein.

4. The method according to claim 1, wherein said protein comprises more than about 30% of the total proteins expressed in the host cell.

5. The method of claim 1, wherein said vector is selected from the group consisting of pET-17b, pET-1 la, pET-24a-d(+) and pET-9a.

6. A method of purifying a recombinantly produced outer membrane meningococcal group B porin protein or fusion protein thereof obtained according to the method of claim 1, comprising: lysing E. coli AompA host cells produced in step of claim 1 to release the protein as insoluble inclusion bodies; washing said insoluble inclusion bodies with a buffer to remove contaminating E. coli cellular proteins; suspending and dissolving said inclusion bodies in an aqueous solution of a denaturant; diluting said solution with a detergent; and purifying said protein by gel filtration and ion exchange chromatography. 4_ Z6/ 58 21/11/97 X034 0 1 0 61 pa 59

7. The method of claim 6, wherein the diluted solution obtained in step has a concentration of less than 10 mg protein/ml.

8. The method of claim 1, wherein said gene codes for a fusion protein which is a mature protein fused to amino acids 1 to 20 or 1 to 22 of the T7 gene 010 capsid protein.

9. The method of claim 1, wherein said gene codes for a mature porin protein.

10. The method of claim 1, wherein said E. coli strain is E co/i BL21 (DE3)AompA.

11. An E. coli AompA host cell that contains a vector which comprises a o* DNA molecule coding for a meningococcal gioup B porin protein or fusion protein thereof operably linked to the T7 promotor of said vector. li

12. The E. coi strain of claim 22, wherein said DNA molecule codes for the mature meningococcal group B class 2 porin protein.

13. The E. col/strain of claim 22, wherein said DNA molecule codes for the mature meningococcal group B class 3 porin protein.

14. The E. coli strain of claim 22, wherein said DNA molecule codes for a fusion protein having the amino acid sequence depicted in figure 4. 27/02/98 L I The E. coli strain of claim 22, wherein said vector is pET-17b.

16. The E. coi AompA host cell of claim 22, wherein said host cell is E Coli strain BL21 (DE3)AompA.

17. E Coli strain BL21(DE3)AompA. Dated this 27th day of February, 1998 THE ROCKEFELLER UNIVERSITY and NORTH AMERICAN VACCINE, INC. Patent Attorneys for the Applicants PETER MAXWELL ASSOCIATES a D r o o o o r o e o or e o 27/02/98 I ~C _CI~-L=LII